WO2012065105A2 - Vaccins contre des flavivirus chimériques - Google Patents

Vaccins contre des flavivirus chimériques Download PDF

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Publication number
WO2012065105A2
WO2012065105A2 PCT/US2011/060436 US2011060436W WO2012065105A2 WO 2012065105 A2 WO2012065105 A2 WO 2012065105A2 US 2011060436 W US2011060436 W US 2011060436W WO 2012065105 A2 WO2012065105 A2 WO 2012065105A2
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flavivirus
virus
chimeric
vector
cell
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PCT/US2011/060436
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WO2012065105A3 (fr
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Thomas Monath
Nikos Vasilakis
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Thomas Monath
Nikos Vasilakis
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2770/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Flaviviruses are members of the genus Flavivirus, which is classified within the family Flaviviridae. Many flaviviruses are significantly pathogenic for humans and other mammals. Two of the most important pathogens of the Flavivirus genus are dengue (serotypes 1, 2, 3 and 4) and yellow fever.
  • Dengue viruses are mosquito-borne viruses and are small (-50 nm), spherical viruses comprising an envelope composed of lipid and two viral proteins (the membrane and envelope proteins) that package and protect the positive-sense single-strand RNA genome and capsid protein.
  • the ⁇ 11 kilobase RNA genome contains a single long open reading frame encoding three structural proteins (the capsid protein, pre-membrane and envelope) and 7 non-structural proteins that are needed for replication but which remain in the infected cell and are not incorporated into the mature virus particle.
  • the pre-membrane protein is truncated during virus assembly to leave the mature membrane protein incorporated into the viral envelope.
  • the non-structural genes encode enzymes, including protease involved in post-translational processing of viral proteins, and helicase and polymerase involved in replication of the viral RNA.
  • enzymes including protease involved in post-translational processing of viral proteins, and helicase and polymerase involved in replication of the viral RNA.
  • At both the 3 ' and 5 ' termini of the viral genome there are short non-coding regions which form structural elements that allow for the polymerase to anchor and initiate replication of the positive- and negative-polarity R A strands during replication.
  • the dengue virus genome organization and function of the encoded viral proteins are reviewed in Lindenbach et al., 2003, Adv Virus Res 59: 23-61, and Perera et al, 2010, Curr Opin Microbiol 11 : 369-77.
  • Dengue is endemic and epidemic in tropical regions of the world, with up to 100 million persons infected every year. (Gubler DJ, 2004, Comp Immunol Microbiol Infect Dis. 27: 319-30; Halstead SB, 2007, Lancet 370: 1644-52). Humans infected with dengue viruses experience febrile illness with severe muscle pains, headache and rash (dengue fever). Severe dengue, also known as dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), is an immunopathological disease that occurs in individuals, mainly children, who sustain sequential infections with different dengue virus serotypes. Therefore, immunity to all four dengue serotypes should be evoked simultaneously by a successful vaccine, as described in detail below.
  • DHF/DSS dengue hemorrhagic fever/dengue shock syndrome
  • the yellow fever virus is in the Flavivirus genus, in the family Flaviviridae.
  • the yellow fever virus is maintained in nature in a transmission cycle involving nonhuman primates and mosquitoes in certain tropical areas of Africa and the Americas. Yellow fever occurs in sporadic human cases and periodically amplifies into epidemics. Other parts of the world, including coastal regions of South America, the Caribbean Islands, and Central and North America, are infested with the mosquito vector capable of transmitting the virus and are therefore considered at risk for introduction and spread of yellow fever epidemics. Illness from the yellow fever virus ranges in severity from a self- limited febrile illness to severe hepatitis and fatal hemorrhagic disease.
  • Unvaccinated humans including both residents of and travelers to yellow fever endemic areas are at significant risk of yellow fever infection when occupational and other activities bring them in contact with infected mosquitoes. There is no specific treatment for yellow fever. Steps to prevent yellow fever include the use of insect repellent, protective clothing, and vaccination with the available, but risky attenuated vaccine.
  • Live, attenuated vaccines for the dengue virus and the yellow fever virus are available and/or in clinical development. There are problems, however, associated with the use of these live, attenuated vaccines.
  • live, attenuated vaccines have been produced from the 17D substrain, but adverse events associated with the attenuated vaccine can lead to a severe infection with the live 17D virus, and serious and fatal adverse neurotropic and viscerotropic events, the latter resembling the severe infection caused by the wild-type yellow fever virus.
  • current investigational vaccines are a mixture of four live viruses (each representing one of the four dengue serotypes).
  • a successful vaccine development strategy is to grow the virus in an acceptable cell culture system, to harvest the virus which is released into the cell culture medium, and to inactivate the virus or produce a virus incapable of replication in mammals, preserving its structure and epitopes.
  • Non-replicating vaccine components in a tetravalent mixture would not interfere with one another, and it is expected that a balanced response to all four serotypes, and high titers of antibody could be evoked using this method.
  • dengue viruses and yellow fever viruses grow to maximum levels of only about 10 7 PFU/mL, 10 to 100-fold (or more) lower than other Flaviviruses against which successful and approved inactivated vaccines have been developed, such as Japanese encephalitis and tick-borne encephalitis. This severely restricts the ability to manufacture inactivated whole virion vaccines against dengue and yellow fever.
  • dengue virus and/or yellow fever virus at high yields (greater than or equal to 10 8 PFU/mL) for preparing whole virion vaccines would be highly desirable.
  • the efficacy of such a dengue or yellow fever vaccine would be expected to be similar to inactivated vaccines against other Flaviviruses, such as Japanese encephalitis (IXIARO® Prescribing information, 2009, Intercell AG) and tick-borne encephalitis (Schondorf et ah, 2007, Vaccine 25: 1470-5).
  • the present invention meets the need for highly-replicating dengue viruses and yellow fever viruses by providing chimeric viral vectors, viruses encoded thereof as well as related vaccines comprising dengue virus or yellow fever -virus components and methods for growing these viruses in cell cultures to levels that allow efficient manufacture of inactivated, whole virion vaccines.
  • the present invention is based, in part, on the discovery that one or more structural proteins from a first fiavivirus with low level of replication in cells can be integrated into a second fiavivirus with a high level of replication in cells, e.g., one or more structural proteins of a first fiavivirus can be used to replace one or more structural proteins of a second fiavivirus.
  • the present invention provides chimeric fiavivirus vectors, chimeric flaviviruses encoded thereof, and vaccines containing chimeric fiavivirus vectors or chimeric flavivirus(es).
  • the present invention also provides methods of vaccination or inducing an immune response using the vaccines of the present invention.
  • the invention provides a chimeric fiavivirus vector encoding a structural protein from a first fiavivirus with a low level of replication in a cell and a backbone from a second fiavivirus with a high level of replication in the cell.
  • the invention provides a chimeric fiavivirus vector encoding an envelope protein from a first fiavivirus with a low level of replication in a cell and one or more nonstructural proteins from a second fiavivirus with a high level of replication in the cell.
  • the structural protein is an envelope protein.
  • the structural protein comprises an envelope protein and a pre -membrane protein.
  • the backbone comprises a capsid protein, the non-structural proteins, and the 3' and 5' non-coding termini.
  • the first fiavivirus with a low level of replication in a cell is selected from the group consisting of dengue virus and yellow fever virus.
  • the first fiavivirus is a dengue virus selected from the group consisting of dengue type 1 (DEN1) virus, dengue type 2 (DEN2) virus, dengue type 3 (DEN3) virus, and dengue type 4 (DEN4) virus.
  • the second fiavivirus with a high level of replication in the cell is a fiavivirus selected from the Rio Bravo taxonomic group.
  • the fiavivirus of the Rio Bravo taxonomic group may be selected from the group consisting of the Rio Bravo (sensu stricto) virus, the Montana Myotis Leukoencephalitis virus, the Dakar Bat virus, the Phnom Penh Bat virus, the Carey Island virus, and the Bukalasa Bat virus.
  • the second flavivirus with a high level of replication in the cell is a flavivirus selected from the Kenya S taxonomic group.
  • the flavivirus of the Philippine S taxonomic group may be selected from the group consisting of the Kenya S virus, the Banzi virus, and the Jugra virus.
  • the second flavivirus with a high level of replication in the cell is a flavivirus selected from the mosquito-associated flaviviruses.
  • the mosquito-associated flavivirus may be selected from the group consisting of the Kamiti River virus, the Culex flavivirus, the Aedes flavivirus, the Nakiwogo virus, the Quang Binh virus, and the Cell Fusing Agent virus.
  • the backbone may be selected from a flavivirus that has been adapted to a cell substrate, e.g., acceptable for use in preparing vaccines for human or veterinary use.
  • the backbone may be selected from a flavivirus that has been adapted to Vero, Madin Darby Canine kidney (MDCK), A549, fetal rhesus lung (FRhL), MRC5, human embryonic kidney (HEK293), primary dog kidney, primary rabbit kidney, xenopus oocytes, or chick embryo cells.
  • the backbone is selected from a flavivirus adapted to a cell substrate such that an increase in yield of the virus is at least about 2.5-fold as compared to the flavivirus before adaptation.
  • the backbone may comprise at least one amino acid modification in a nonstructural protein.
  • the non-structural protein is NS 1.
  • the NS 1 protein may comprise a substitution at an amino acid position corresponding to the proline 315 residue of the Rio Bravo virus NS1 (SEQ ID NO: 1).
  • the proline 315 residue of the NS1 protein is replaced with a serine residue.
  • the non-structural protein is NS3.
  • the NS3 protein may comprise a substitution at an amino acid position corresponding to the isoleucine 555 residue of the Rio Bravo virus NS3 (SEQ ID NO: 2).
  • the isoleucine 555 residue of the NS3 protein is replaced with a threonine residue.
  • the chimeric flavivirus vector may comprise at least one nucleotide deletion in the 3' non-coding region (NCR).
  • the 3' non-coding region may comprise a nucleotide deletion at a nucleotide corresponding to the thymine at position 10692 of the Rio Bravo virus genome.
  • the chimeric flavivirus vector may further comprise a signal sequence at the 3' end of the capsid gene, e.g., a target for a host cell furin enzyme which cleaves the translated open reading frame at that site.
  • the chimeric flavivirus vector may be within a plasmid comprising at least one cytomegalovirus (CMV) promoter, e.g., operably linked to the chimeric flavivirus vector.
  • CMV cytomegalovirus
  • the chimeric flavivirus vector may be within a plasmid expressing at least one hepatitis ⁇ virus ribozyme.
  • the chimeric flavivirus vector may be within a plasmid comprising at least one SV40 polyadenylation site.
  • the chimeric flavivirus vector may comprise one or more intron sequences.
  • the chimeric flavivirus vector may comprise an intron at the junction of the envelope (E) and the non- structural gene 1 (NS1).
  • the chimeric flavivirus vector may comprise an intron immediately after a nucleotide corresponding to position 9742 of the Rio Bravo virus genome.
  • the chimeric flavivirus vector encodes a chimeric flavivirus capable of replicating in mammalian cells to at least about 10 8 PFU/mL. In a further embodiment, the chimeric flavivirus vector encodes a chimeric flavivirus capable of replicating in mammalian cells to at least about 10 9 PFU/mL. In additional embodiments, the chimeric flavivirus vector encodes a chimeric flavivirus capable of replicating in insect cells, but not capable of replicating in human cells.
  • the invention provides a chimeric flavivirus vector encoding a structural protein from a first flavivirus with a low level of replication in a cell and a backbone from a second flavivirus with a high level of replication in the cell, wherein the structural protein is an envelope protein and the backbone comprises the capsid protein, the non-structural proteins and the 3' and 5' noncoding termini, and wherein the second flavivirus is a flavivirus that does not cause cytopathic effect (CPE) when growing in a cell culture.
  • the second flavivirus allows more than one harvesting of a cell culture fluid from a cell culture when growing in the cell culture.
  • the first flavivirus with a low level of replication in a cell is selected from the group consisting of dengue virus and yellow fever virus.
  • the first flavivirus is a dengue virus selected from the group consisting of dengue type 1 (DEN1) virus, dengue type 2 (DEN2) virus, dengue type 3 (DEN3) virus, and dengue type 4 (DEN4) virus.
  • the second flavivirus is a flavivirus selected from the Rio Bravo taxonomic group.
  • the flavivirus of the Rio Bravo taxonomic group may be selected from the group consisting of the Rio Bravo (sensu stricto) virus, the Montana Myotis Leukoencephalitis virus, the Dakar Bat virus, the Phnom Penh Bat virus, the Carey Island virus, and the Bukalasa Bat virus.
  • the second flavivirus with a high level of replication in the cell is a flavivirus selected from the mosquito-associated flaviviruses.
  • the mosquito-associated flavivirus may be selected from the group consisting of the Kamiti River virus, the Culex flavivirus, the Aedes flavivirus, the Nakiwogo virus, the Quang Binh virus, and the Cell Fusing Agent virus.
  • the second flavivirus may be selected from a flavivirus that has been adapted to a cell substrate.
  • the second flavivirus may be selected from a flavivirus that has been adapted to Vera, Madin Darby Canine kidney (MDCK), A549, fetal rhesus lung (FRhL), MRC5, human embryonic kidney (HEK293), primary dog kidney, primary rabbit kidney, xenopus oocytes, or chick embryo cells.
  • the backbone is selected from a flavivirus adapted to a cell substrate such that an increase in yield of the virus is at least about 2.5 -fold as compared to the flavivirus before adaptation.
  • the second flavivirus may comprise at least one amino acid modification in a non-structural protein.
  • the non-structural protein is NS 1.
  • the NS 1 protein may comprise a substitution at an amino acid position corresponding to the proline 315 residue of the Rio Bravo virus NS1 (SEQ ID NO: 1).
  • the proline 315 residue of the NS1 protein is replaced with a serine residue.
  • the non-structural protein is NS3.
  • the NS3 protein may comprise a substitution at an amino acid position corresponding to the isoleucine 555 residue of the Rio Bravo virus NS3 (SEQ ID NO: 2).
  • the isoleucine 555 residue of the NS3 protein is replaced with a threonine residue.
  • the chimeric flavivirus vector may comprise at least one nucleotide deletion in the 3' non-coding region (NCR).
  • the 3' non-coding region may comprise a nucleotide deletion at a nucleotide corresponding to the thymine at position 10692 of the Rio Bravo virus genome.
  • the chimeric fiavivirus vector may further comprise a signal sequence at the 3' end of the capsid gene.
  • the chimeric fiavivirus vector may be within a plasmid comprising at least one cytomegalovirus (CMV) promoter, e.g., operably linked to the chimeric fiavivirus vector.
  • CMV cytomegalovirus
  • the chimeric fiavivirus vector may be within a plasmid expressing at least one hepatitis ⁇ virus ribozyme.
  • the chimeric fiavivirus vector may be within a plasmid comprising at least one SV40 polyadenylation site.
  • the chimeric fiavivirus vector may comprise one or more intron sequences.
  • the chimeric fiavivirus vector may comprise an intron at the junction of the envelope (E) and the non- structural gene 1 (NS1).
  • the chimeric fiavivirus vector may comprise an intron immediately after a nucleotide corresponding to position 9742 of the Rio Bravo virus genome.
  • the chimeric fiavivirus vector encodes a chimeric fiavivirus capable of replicating in mammalian cells to at least about 10 8 PFU/mL. In a further embodiment, the chimeric fiavivirus vector encodes a chimeric fiavivirus capable of replicating in mammalian cells to at least about 10 9 PFU/mL. In additional embodiments, the chimeric fiavivirus vector encodes a chimeric fiavivirus capable of replicating in insect cells, but not capable of replicating in human cells.
  • the invention provides a chimeric fiavivirus vector encoding a structural protein from a first fiavivirus with a low level of replication in a cell and a backbone from a second fiavivirus with a high level of replication in the cell, wherein the structural protein is an envelope protein and the backbone comprises the capsid protein, the non-structural proteins and the 3' and 5' noncoding termini, and wherein the second fiavivirus is a fiavivirus that does not cause an elevation in extracellular DNA release as compared to a mock infection when growing in a cell culture, and as measured at day 3 postinfection.
  • the first fiavivirus with a low level of replication in a cell is selected from the group consisting of dengue virus and yellow fever virus.
  • the first fiavivirus is a dengue virus selected from the group consisting of dengue type 1 (DEN1) virus, dengue type 2 (DEN2) virus, dengue type 3 (DEN3) virus, and dengue type 4 (DEN4) virus.
  • the second flavivirus with a high level of replication in the cell is a flavivirus selected from the Rio Bravo taxonomic group.
  • the flavivirus of the Rio Bravo taxonomic group may be selected from the group consisting of the Rio Bravo (sensu stricto) virus, the Montana Myotis Leukoencephalitis virus, the Dakar Bat virus, the Phnom Penh Bat virus, the Carey Island virus, and the Bukalasa Bat virus.
  • the second flavivirus with a high level of replication in the cell is a flavivirus selected from the mosquito-associated flaviviruses.
  • the mosquito-associated flavivirus may be selected from the group consisting of the Kamiti River virus, the Culex flavivirus, the Aedes flavivirus, the Nakiwogo virus, the Quang Binh virus, and the Cell Fusing Agent virus.
  • the second flavivirus may be selected from a flavivirus that has been adapted to a cell substrate.
  • the second flavivirus may be selected from a flavivirus that has been adapted to Vera, Madin Darby Canine kidney (MDCK), A549, fetal rhesus lung (FRhL), MRC5, human embryonic kidney (HEK293), primary dog kidney, primary rabbit kidney, xenopus oocytes, or chick embryo cells.
  • the backbone is selected from a flavivirus adapted to a cell substrate such that an increase in yield of the virus is at least about 2.5 -fold as compared to the flavivirus before adaptation.
  • the second flavivirus may comprise at least one amino acid modification in a non-structural protein.
  • the non-structural protein is NS 1.
  • the NS 1 protein may comprise a substitution at an amino acid position corresponding to the proline 315 residue of the Rio Bravo virus NS1 (SEQ ID NO: 1).
  • the proline 315 residue of the NS1 protein is replaced with a serine residue.
  • the non-structural protein is NS3.
  • the NS3 protein may comprise a substitution at an amino acid position corresponding to the isoleucine 555 residue of the Rio Bravo virus NS3 (SEQ ID NO: 2).
  • the isoleucine 555 residue of the NS3 protein is replaced with a threonine residue.
  • the chimeric flavivirus vector may comprise at least one nucleotide deletion in the 3' non-coding region (NCR).
  • the 3' non-coding region may comprise a nucleotide deletion at a nucleotide corresponding to the thymine at position 10692 of the Rio Bravo virus genome.
  • the chimeric flavivirus vector may further comprise a signal sequence at the 3' end of the capsid gene.
  • the chimeric flavivirus vector may be within a plasmid comprising at least one cytomegalovirus (CMV) promoter, e.g., operably linked to the chimeric flavivirus vector.
  • CMV cytomegalovirus
  • the chimeric flavivirus vector may be within a plasmid expressing at least one hepatitis ⁇ virus ribozyme.
  • the chimeric flavivirus vector may be within a plasmid comprising at least one SV40 polyadenylation site.
  • the chimeric flavivirus vector may comprise one or more intron sequences.
  • the chimeric flavivirus vector may comprise an intron at the junction of the envelope (E) and the non- structural gene 1 (NS1).
  • the chimeric flavivirus vector may comprise an intron immediately after a nucleotide corresponding to position 9742 of the Rio Bravo virus genome.
  • the chimeric flavivirus vector encodes a chimeric flavivirus capable of replicating in mammalian cells to at least about 10 8 PFU/mL. In a further embodiment, the chimeric flavivirus vector encodes a chimeric flavivirus capable of replicating in mammalian cells to at least about 10 9 PFU/mL. In additional embodiments, the chimeric flavivirus vector encodes a chimeric flavivirus capable of replicating in insect cells, but not capable of replicating in human cells.
  • the invention provides a chimeric flavivirus encoded by a chimeric flavivirus vector.
  • the chimeric flavivirus is encoded by a chimeric flavivirus vector, wherein the chimeric flavivirus vector encodes a structural protein from a first flavivirus with a low level of replication in a cell and a backbone from a second flavivirus with a high level of replication in the cell.
  • the chimeric flavivirus is encoded by a chimeric flavivirus vector, wherein the chimeric flavivirus vector encodes an envelope protein from a first flavivirus with a low level of replication in a cell and one or more non-structural proteins from a second flavivirus with a high level of replication in the cell.
  • the structural protein is an envelope protein.
  • the structural protein comprises an envelope protein and a pre -membrane protein.
  • the backbone comprises a capsid protein, the non-structural proteins, and the 3' and 5' non-coding termini.
  • the first flavivirus with a low level of replication in a cell is selected from the group consisting of dengue virus and yellow fever virus.
  • the first flavivirus is a dengue virus selected from the group consisting of dengue type 1 (DEN1) virus, dengue type 2 (DEN2) virus, dengue type 3 (DEN3) virus, and dengue type 4 (DEN4) virus.
  • the second flavivirus with a high level of replication in the cell is a flavivirus selected from the Rio Bravo taxonomic group.
  • the flavivirus of the Rio Bravo taxonomic group may be selected from the group consisting of the Rio Bravo (sensu stricto) virus, the Montana Myotis Leukoencephalitis virus, the Dakar Bat virus, the Phnom Penh Bat virus, the Carey Island virus, and the Bukalasa Bat virus.
  • the second flavivirus with a high level of replication in the cell is a flavivirus selected from the Kenya S taxonomic group.
  • the flavivirus of the Kenya S taxonomic group may be selected from the group consisting of the Kenya S virus, the Banzi virus, and the Jugra virus.
  • the second flavivirus with a high level of replication in the cell is a flavivirus selected from the mosquito-associated flaviviruses.
  • the mosquito-associated flavivirus may be selected from the group consisting of the Kamiti River virus, the Culex flavivirus, the Aedes flavivirus, the Nakiwogo virus, the Quang Binh virus, and the Cell Fusing Agent virus.
  • the backbone may be selected from a flavivirus that has been adapted to a cell substrate.
  • the backbone may be selected from a flavivirus that has been adapted to Vera, Madin Darby Canine kidney (MDCK), A549, fetal rhesus lung (FRhL), MRC5, human embryonic kidney (HEK293), primary dog kidney, primary rabbit kidney, xenopus oocytes, or chick embryo cells.
  • the backbone is selected from a flavivirus adapted to a cell substrate such that an increase in yield of the virus is at least about 2.5 -fold as compared to the flavivirus before adaptation.
  • the backbone may comprise at least one amino acid modification in a nonstructural protein.
  • the non-structural protein is NS 1.
  • the NS 1 protein may comprise a substitution at an amino acid position corresponding to the proline 315 residue of the Rio Bravo virus NS1 (SEQ ID NO: 1).
  • the proline 315 residue of the NS1 protein is replaced with a serine residue.
  • the non-structural protein is NS3.
  • the NS3 protein may comprise a substitution at an amino acid position corresponding to the isoleucine 555 residue of the Rio Bravo virus NS3 (SEQ ID NO: 2).
  • the isoleucine 555 residue of the NS3 protein is replaced with a threonine residue.
  • the chimeric flavivirus vector may comprise at least one nucleotide deletion in the 3' non-coding region (NCR).
  • the 3' non-coding region may comprise a nucleotide deletion at a nucleotide corresponding to the thymine at position 10692 of the Rio Bravo virus genome.
  • the chimeric flavivirus vector may further comprise a signal sequence at the 3' end of the capsid gene.
  • the chimeric flavivirus vector may be within a plasmid comprising at least one cytomegalovirus (CMV) promoter, e.g., operably linked to the chimeric flavivirus vector.
  • CMV cytomegalovirus
  • the chimeric flavivirus vector may be within a plasmid expressing at least one hepatitis ⁇ virus ribozyme.
  • the chimeric flavivirus vector may be within a plasmid comprising at least one SV40 polyadenylation site.
  • the chimeric flavivirus vector may comprise one or more intron sequences.
  • the chimeric flavivirus vector may comprise an intron at the junction of the envelope (E) and the non- structural gene 1 (NS1).
  • the chimeric flavivirus vector may comprise an intron immediately after a nucleotide corresponding to position 9742 of the Rio Bravo virus genome.
  • the chimeric flavivirus vector encodes a chimeric flavivirus capable of replicating in mammalian cells to at least about 10 8 PFU/mL. In a further embodiment, the chimeric flavivirus vector encodes a chimeric flavivirus capable of replicating in mammalian cells to at least about 10 9 PFU/mL. In additional embodiments, the chimeric flavivirus vector encodes a chimeric flavivirus capable of replicating in insect cells, but not capable of replicating in human cells.
  • the invention provides an inactivated chimeric flavivirus encoded by a chimeric flavivirus vector described herein or alternatively derived from a chimeric flavivirus comprising a structural protein, e.g., envelope protein and optionally a membrane protein from a first flavivirus with a low level of replication in a cell and 1) a capsid protein from a second flavivirus with a high level of replication in the cell, 2) an RNA genome comprising a sequence encoding one or more or all of the non-structural proteins, e.g., NSl, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 from a second flavivirus with a high level of replication in the cell, or 3) an RNA genome with both 3' and 5' termini of the genome from a second flavivirus with a high level of replication in the cell, or a combination thereof.
  • a structural protein e.g., envelope protein and optionally a membrane protein from
  • the chimeric flavivirus e.g., infectious viral particles may be inactivated with a method selected from the group consisting of chemical inactivation, high pressure inactivation, ultraviolet radiation, and gamma radiation.
  • the chimeric flavivirus is inactivated using chemical inactivation.
  • the method of chemical inactivation may comprise exposure of the flavivirus to one or more agents selected from the group consisting of ⁇ -propiolactone, formalin, aziridines, hydrogen peroxide, organic solvents, and ascorbic acid.
  • the invention provides a vaccine comprising an inactivated chimeric flavivirus described herein.
  • the vaccine comprises an inactivated chimeric flavivirus, wherein the inactivated chimeric flavivirus is derived from a chimeric flavivirus vector that encodes a structural protein from a first flavivirus with a low level of replication in a cell and a backbone from a second flavivirus with a high level of replication in the cell.
  • the structural protein is an envelope protein.
  • the structural protein comprises an envelope protein and a pre-membrane protein.
  • the backbone comprises a capsid protein, the non-structural proteins, and the 3' and 5' non-coding termini.
  • the first flavivirus with a low level of replication in a cell is selected from the group consisting of dengue virus and yellow fever virus.
  • the first flavivirus is a dengue virus selected from the group consisting of dengue type 1 (DEN1) virus, dengue type 2 (DEN2) virus, dengue type 3 (DEN3) virus, and dengue type 4 (DEN4) virus.
  • the second flavivirus with a high level of replication in the cell is a flavivirus selected from the Rio Bravo taxonomic group.
  • the flavivirus of the Rio Bravo taxonomic group may be selected from the group consisting of the Rio Bravo (sensu stricto) virus, the Montana Myotis Leukoencephalitis virus, the Dakar Bat virus, the Phnom Penh Bat virus, the Carey Island virus, and the Bukalasa Bat virus.
  • the second flavivirus with a high level of replication in the cell is a flavivirus selected from the Kenya S taxonomic group.
  • the flavivirus of the Kenya S taxonomic group may be selected from the group consisting of the Kenya S virus, the Banzi virus, and the Jugra virus.
  • the second flavivirus with a high level of replication in the cell is a flavivirus selected from the mosquito-associated flaviviruses.
  • the mosquito-associated flavivirus may be selected from the group consisting of the Kamiti River virus, the Culex flavivirus, the Aedes flavivirus, the Nakiwogo virus, the Quang Binh virus, and the Cell Fusing Agent virus.
  • the backbone may be selected from a flavivirus that has been adapted to a cell substrate.
  • the backbone may be selected from a flavivirus that has been adapted to Vera, Madin Darby Canine kidney (MDCK), A549, fetal rhesus lung (FRhL), MRC5, human embryonic kidney (HEK293), primary dog kidney, primary rabbit kidney, xenopus oocytes, or chick embryo cells.
  • the backbone is selected from a flavivirus adapted to a cell substrate such that an increase in yield of the virus is at least about 2.5 -fold as compared to the flavivirus before adaptation.
  • the backbone may comprise at least one amino acid modification in a nonstructural protein.
  • the non-structural protein is NS 1.
  • the NS 1 protein may comprise a substitution at an amino acid position corresponding to the proline 315 residue of the Rio Bravo virus NSl (SEQ ID NO: 1).
  • the proline 315 residue of the NSl protein is replaced with a serine residue.
  • the non-structural protein is NS3.
  • the NS3 protein may comprise a substitution at an amino acid position corresponding to the isoleucine 555 residue of the Rio Bravo virus NS3 (SEQ ID NO: 2).
  • the isoleucine 555 residue of the NS3 protein is replaced with a threonine residue.
  • the chimeric flavivirus vector may comprise at least one nucleotide deletion in the 3' non-coding region (NCR).
  • the 3' non-coding region may comprise a nucleotide deletion at a nucleotide corresponding to the thymine at position 10692 of the Rio Bravo virus genome.
  • the chimeric flavivirus vector may further comprise a signal sequence at the 3' end of the capsid gene.
  • the chimeric flavivirus vector may be within a plasmid comprising at least one cytomegalovirus (CMV) promoter, e.g., operably linked to the chimeric flavivirus vector.
  • CMV cytomegalovirus
  • the chimeric flavivirus vector may be within a plasmid expressing at least one hepatitis ⁇ virus ribozyme.
  • the chimeric flavivirus vector may be within a plasmid comprising at least one SV40 polyadenylation site.
  • the chimeric flavivirus vector may comprise one or more intron sequences.
  • the chimeric flavivirus vector may comprise an intron at the junction of the envelope (E) and the non- structural gene 1 (NSl).
  • the chimeric flavivirus vector may comprise an intron immediately after a nucleotide corresponding to position 9742 of the Rio Bravo virus genome.
  • the chimeric flavivirus vector encodes a chimeric flavivirus capable of replicating in mammalian cells to at least about 10 8 PFU/mL. In a further embodiment, the chimeric flavivirus vector encodes a chimeric flavivirus capable of replicating in mammalian cells to at least about 10 9 PFU/mL.
  • the invention provides a vaccine comprising an inactivated chimeric flavivirus.
  • the inactivated chimeric flavivirus is derived from a chimeric flavivirus comprising a structural protein, e.g., envelope protein or membrane protein or both from a first flavivirus with a low level of replication in a cell and 1) a capsid protein from a second flavivirus with a high level of replication in the cell, 2) an RNA genome comprising a sequence encoding one or more or all of the non-structural proteins, e.g., NSl, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 from a second flavivirus with a high level of replication in the cell, or 3) an RNA genome with both 3' and 5' termini of the genome from a second flavivirus with a high level of replication in the cell, or a combination thereof.
  • a structural protein e.g., envelope protein or membrane protein or both from a first
  • the first flavivirus with a low level of replication in a cell is selected from the group consisting of dengue virus and yellow fever virus.
  • the first fiavivirus is a dengue virus selected from the group consisting of dengue type 1 (DEN1) virus, dengue type 2 (DEN2) virus, dengue type 3 (DEN3) virus, and dengue type 4 (DEN4) virus.
  • the second fiavivirus with a high level of replication in the cell is a fiavivirus selected from the Rio Bravo taxonomic group.
  • the fiavivirus of the Rio Bravo taxonomic group may be selected from the group consisting of the Rio Bravo (sensu stricto) virus, the Montana Myotis Leukoencephalitis virus, the Dakar Bat virus, the Phnom Penh Bat virus, the Carey Island virus, and the Bukalasa Bat virus.
  • the second fiavivirus with a high level of replication in the cell is a fiavivirus selected from the Kenya S taxonomic group.
  • the fiavivirus of the Kenya S taxonomic group may be selected from the group consisting of the Kenya S virus, the Banzi virus, and the Jugra virus.
  • the second fiavivirus with a high level of replication in the cell is a fiavivirus selected from the mosquito-associated flaviviruses.
  • the mosquito-associated fiavivirus may be selected from the group consisting of the Kamiti River virus, the Culex fiavivirus, the Aedes fiavivirus, the Nakiwogo virus, the Quang Binh virus, and the Cell Fusing Agent virus.
  • the second fiavivirus with a high level of replication in the cell is a West Nile virus.
  • the second fiavivirus may be selected from a fiavivirus that has been adapted to a cell substrate.
  • the second fiavivirus may be selected from a fiavivirus that has been adapted to Vera, Madin Darby Canine kidney (MDCK), A549, fetal rhesus lung (FRhL), MRC5, human embryonic kidney (HEK293), primary dog kidney, primary rabbit kidney, xenopus oocytes, or chick embryo cells.
  • the backbone is selected from a fiavivirus adapted to a cell substrate such that an increase in yield of the virus is at least about 2.5 -fold as compared to the fiavivirus before adaptation.
  • the second fiavivirus may comprise at least one amino acid modification in a non-structural protein.
  • the non-structural protein is NS 1.
  • the NS 1 protein may comprise a substitution at an amino acid position corresponding to the proline 315 residue of the Rio Bravo virus NSl (SEQ ID NO: 1).
  • the proline 315 residue of the NSl protein is replaced with a serine residue.
  • the non-structural protein is NS3.
  • the NS3 protein may comprise a substitution at an amino acid position corresponding to the isoleucine 555 residue of the Rio Bravo virus NS3 (SEQ ID NO: 2).
  • the isoleucine 555 residue of the NS3 protein is replaced with a threonine residue.
  • the chimeric flavivirus is capable of replicating in mammalian cells to at least about 10 8 PFU/mL. In a further embodiment, the chimeric flavivirus is capable of replicating in mammalian cells to at least about 10 9 PFU/mL. In additional embodiments, the chimeric fiavivirus is capable of replicating in insect cells, but not capable of replicating in human cells.
  • vaccines of the invention may additionally comprise an adjuvant.
  • a vaccine described herein may comprise an adjuvant selected from the group consisting of aluminum hydroxide, MF59, saponin, lipid A, iscomatrix, and immunostimulatory oligonucleotides.
  • the invention provides a vaccine comprising a live, chimeric flavivirus described herein.
  • the vaccine comprises a live, chimeric fiavivirus encoded by the chimeric flavivirus described herein and/or comprising a structural protein from a first fiavivirus with a low level of replication in a cell and 1) a capsid protein from a second flavivirus with a high level of replication in the cell, 2) an RNA genome comprising a sequence encoding one or more or all of the non-structural proteins, e.g., NSl, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 from a second flavivirus with a high level of replication in the cell, or 3) an RNA genome with both 3' and 5' termini of the genome from a second fiavivirus with a high level of replication in the cell, or a combination thereof.
  • the structural protein is an envelope protein. In another embodiment, the structural protein comprises an envelope protein and a pre -membrane protein. In one embodiment, the first flavivirus with a low level of replication in a cell is selected from the group consisting of dengue virus and yellow fever virus. In another embodiment, the first flavivirus is a dengue virus selected from the group consisting of dengue type 1 (DEN1) virus, dengue type 2 (DEN2) virus, dengue type 3 (DEN3) virus, and dengue type 4 (DEN4) virus.
  • DEN1 dengue type 1
  • DE3 dengue type 2
  • DEN4 dengue type 4
  • the second flavivirus with a high level of replication in the cell is a flavivirus selected from the mosquito-associated flaviviruses.
  • the mosquito-associated flavivirus may be selected from the group consisting of the Kamiti River virus, the Culex flavivirus, the Aedes flavivirus, the Nakiwogo virus, the Quang Binh virus, and the Cell Fusing Agent virus.
  • the backbone may be selected from a flavivirus that has been adapted to a cell substrate.
  • the backbone may be selected from a flavivirus that has been adapted to C6/36 Aedes albopictus mosquito cells, u4.4 cells, High FiveTM cells, Schneider's Drosophila cell line 2, Spodoptera frugiperda SF9 cells, Anopheles albimanus cells, Anopheles gambiae cells, Culex tar sails cells, Phlebotomus papatasi cells, and C7-10 cells.
  • the backbone is selected from a flavivirus adapted to a cell substrate such that an increase in yield of the virus is at least about 2.5-fold as compared to the flavivirus before adaptation.
  • the chimeric flavivirus is capable of replicating in mammalian cells to at least about 10 8 PFU/mL. In a further embodiment, the chimeric flavivirus is capable of replicating in mammalian cells to at least about 10 9 PFU/mL. In additional embodiments, the chimeric flavivirus vector encodes a chimeric flavivirus capable of replicating in insect cells, but not capable of replicating in human cells.
  • the invention provides vaccines comprising one or more chimeric flavivirus vectors described herein.
  • the chimeric flavivirus vector encodes a structural protein from a first flavivirus with a low level of replication in a cell and a backbone from a second flavivirus with a high level of replication in the cell.
  • the chimeric flavivirus vector encodes an envelope protein from a first flavivirus with a low level of replication in a cell and one or more non-structural proteins from a second flavivirus with a high level of replication in the cell.
  • the chimeric flavivirus vector encodes a structural protein from a first flavivirus with a low level of replication in a cell and a backbone from a second flavivirus with a high level of replication in the cell, wherein the structural protein is an envelope protein and the backbone comprises the capsid protein, the non-structural proteins and the 3' and 5' noncoding termini, and wherein the second flavivirus is a flavivirus does not cause cytopathic effect (CPE) when growing in a cell culture.
  • CPE cytopathic effect
  • the chimeric flavivirus vector encodes a structural protein from a first flavivirus with a low level of replication in a cell and a backbone from a second flavivirus with a high level of replication in the cell, wherein the structural protein is an envelope protein and the backbone comprises the capsid protein, the non-structural proteins and the 3' and 5' noncoding termini, and wherein the second flavivirus is a flavivirus that does not cause an elevation in extracellular DNA release as compared to a mock infection when growing in a cell culture and as measured at day 3 post-infection.
  • the vaccine may be formulated for parenteral or mucosal, e.g., oral administration.
  • the invention provides a method for inducing an immune response in a subject.
  • the method comprises administration of a chimeric flavivirus encoded by a chimeric flavivirus vector described herein.
  • the chimeric flavivirus is an inactivated chimeric flavivirus.
  • the chimeric flavivirus is a live chimeric flavivirus.
  • the inactivated chimeric flavivirus is administered in conjunction with an adjuvant.
  • the method comprises administration of a chimeric flavivirus vector described herein.
  • the chimeric flavivirus vector is administered in conjunction with an adjuvant.
  • the invention provides a method for inducing an immune response to a dengue virus in a subject.
  • the invention provides a method for inducing an immune response to a yellow fever virus in a subject.
  • the invention provides a method for vaccination of a subject.
  • the method comprises administration of a chimeric flavivirus encoded by a chimeric flavivirus vector described herein.
  • the chimeric flavivirus is an inactivated chimeric flavivirus.
  • the chimeric flavivirus is a live chimeric flavivirus.
  • the inactivated flavivirus is administered in conjunction with an adjuvant.
  • the method comprises administration of a chimeric flavivirus vector described herein.
  • the chimeric flavivirus vector is administered in conjunction with an adjuvant.
  • the invention provides a method for vaccination of a subject to prevent infection with the dengue virus.
  • the invention provides a method for vaccination of a subject to prevent infection with the yellow fever virus.
  • Figure 1 is a diagram which illustrates the phylogenetic relationships of members of the Flavivirus genus. Members of the Rio Bravo taxonomic group, the Philippine S taxonomic group, and the West Nile virus are circled.
  • Figure 2A illustrates the growth of selected flaviviruses in Vera cells (P2, no adaptation).
  • Figure 2B illustrates the (Top) Banzi virus on day 3, exhibiting 4+ cytopathic effects (CPE) and the (Bottom) Rio Bravo virus on day 5, exhibiting minimal cytopathic effects (CPE).
  • Figure 3 illustrates the growth kinetics of the Rio Bravo virus before passage 1 (PI) and after adaptation by serial passage (P10).
  • Figure 4 illustrates the growth kinetics (Vera WHO 10-87 cells) of small plaque versus large plaque population of Rio Bravo virus. The plaque populations differed in genomic sequence as described in the text.
  • Figure 5 illustrates a comparison of growth kinetics of dengue-2 strains belonging to Lineage I and II isolated from humans in Peru.
  • Figure 6 illustrates the growth kinetics of individual dengue-2 strains in Vera cells. Some strains (in Lineage II) grew to relatively high titer (>10 6 PFU/mL). Strain FPI00174 was selected as a donor strain for construction of chimeric virus.
  • Figures 7 A through 7E illustrate a cloning strategy for the construction of a chimeric fiavivirus vector comprising a Rio Bravo virus backbone and a structural protein from a dengue virus.
  • the present invention is based, in part, on the discovery that one or more structural proteins from a first fiavivirus with low level of replication in cells can be integrated into a second fiavivirus with a high level of replication in cells, e.g., one or more structural proteins of a first fiavivirus can be used to replace one or more structural proteins of a second fiavivirus.
  • the present invention provides chimeric fiavivirus vectors, chimeric flaviviruses encoded thereof, and vaccines containing chimeric fiavivirus vectors or chimeric flavivirus(es).
  • the present invention also provides methods of vaccination or inducing an immune response using the vaccines of the present invention.
  • the invention provides a chimeric fiavivirus vector encoding a structural protein from a first fiavivirus with a low level of replication in a cell and a backbone from a second fiavivirus with a high level of replication in the cell.
  • chimeric fiavivirus vector refers to any polynucleotide containing or comprising the desired nucleotide sequence, e.g., the nucleotide sequence encoding the chimeric fiavivirus of the present invention.
  • the polynucleotide can be DNA, RNA, cDNA and can include naturally existing nucleotides or any modified nucleotides.
  • the polynucleotide can be within any suitable construct, e.g., for cloning, infection, replication, expression, etc.
  • the polynucleotide is within a plasmid, e.g., for cloning and/or infection.
  • the polynucleotide is within a viral genome.
  • the polypeptide is independent of any construct.
  • a low level of replication in a cell as it refers to a fiavivirus' ability to replicate in a cell, relates to a wild-type fiavivirus which replicates to maximum levels of less than about 10 6 , 10 7 or 10 8 plaque-forming units (PFUs)/mL in cell culture.
  • PFUs plaque-forming units
  • flaviviruses with a low level of replication in a cell include dengue viruses and yellow fever viruses.
  • a high level of replication in a cell as it refers to a flavivirus' ability to replicate in a cell, relates to a wild-type flavivirus which replicates to maximum levels of at least about 10 8 , 10 9 , or 10 10 plaque-forming units (PFUs)/mL in cell culture.
  • flaviviruses with a high level of replication in a cell include West Nile viruses, Japanese encephalitis viruses, tick-borne encephalitis viruses, flaviviruses of the Rio Bravo taxonomic group, flaviviruses of the Philippine S taxonomic group, and mosquito-associated flaviviruses.
  • a backbone refers to a structural component of a virus genome. In some embodiments, it includes sequences encoding one or more or all non-structural components of a virus genome, e.g., NS1, NS2A, NS2B, NS3, NS4, NS4B, and NS5. In some other embodiments, it includes sequences encoding all non-structural components as well as a capsid protein. In some other embodiments, it includes sequences encoding all nonstructural components, capsid protein as well as 3' and 5' non-coding termini of a virus. In some other embodiments, it includes sequences encoding all components of a virus other than its envelope protein and optionally the membrane protein.
  • it includes naturally existing sequences, modified sequences with natural or non-natural nucleotides.
  • it includes sequences derived from the naturally existing sequences, e.g., it includes sequences found in naturally existing sequences with mutations such as deletions, additions, substitutions so long as products encoded thereof function equivalently or better than the products encoded by naturally existing sequences.
  • the invention is directed, in part, to methods of growing high levels of a flavivirus having the envelope protein (E) (containing protective, neutralizing epitopes), fragments thereof, variants thereof, or derivatives thereof and optionally membrane protein, fragments thereof, variants thereof, or derivatives thereof from a first flavivirus with a low level of replication in a cell in cell culture which can either be rendered safe (non-replicating) by chemical inactivation, or by use of a virus incapable of replicating in humans.
  • the methods of the invention employ the construction of a chimeric flavivirus vector that comprises a sequence encoding the envelope and optionally pre-membrane protein of a first flavivirus with a low level of replication in a cell.
  • the envelope and optionally pre-membrane protein of a first flavivirus when become part of the chimeric flavivirus contains epitopes in their natural conformation and the chimeric flavivirus is capable of replicating to a titer in acceptable cell cultures sufficient for manufacture of an inactivated vaccine.
  • the chimeric flavivirus vector encodes a structural protein from a first flavivirus with a low level of replication in a cell.
  • the structural protein is an envelope (E) protein, fragment thereof, variant thereof, or derivative thereof.
  • the chimeric flavivirus vector encodes an envelope protein, fragment thereof, variant thereof, or derivative thereof, and a pre-membrane protein, fragment thereof, variant thereof, or derivative thereof from a first flavivirus with a low level of replication in a cell.
  • the envelope protein, fragment thereof, variant thereof, or derivative thereof and the pre-membrane protein, fragment thereof, variant thereof, or derivative thereof are from the same flavivirus with a low level of replication in a cell.
  • the envelope protein, fragment thereof, variant thereof, or derivative thereof, and the pre-membrane protein, fragment thereof, variant thereof, or derivative thereof are from a different flavivirus with a low level of replication in a cell, e.g., the pre-membrane protein, fragment thereof, variant thereof, or derivative thereof is from a second flavivirus with a high level of replication in a cell.
  • the disclosure identifies specific genes (e.g., envelope, pre- membrane, non-structural, and/or capsid genes) useful in the compositions and methods of the disclosure.
  • genes e.g., envelope, pre- membrane, non-structural, and/or capsid genes
  • absolute identity to such genes is not necessary.
  • changes in a particular gene or polynucleotide comprising a sequence encoding a polypeptide e.g., an envelope and optionally a pre-membrane polypeptide
  • Such changes comprise conservative mutations and silent mutations.
  • Such modified or mutated polynucleotides and polypeptides can be screened for expression of a functional immunogenic polypeptide using methods known in the art.
  • polynucleotides which encode substantially the same or functionally equivalent polypeptides can also be used to clone and express the polynucleotides encoding such polypeptides.
  • the coding regions encoding fiavivirus polypeptides or fragments, variants, or derivatives thereof may be codon optimized.
  • the coding regions encoding a fiavivirus envelope and/or pre -membrane polypeptide or fragment, variant, or derivative thereof are codon optimized Codon optimization is carried out by the methods well known in the art, for example, in certain embodiments codon-optimized coding regions encoding polypeptides of fiavivirus, or nucleic acid fragments of such coding regions encoding fragments, variants, or derivatives thereof are optimized according to the codon usage of the particular host cell.
  • the host cell is selected from the group consisting of Vero, Madin Darby Canine kidney (MDCK), A549, fetal rhesus lung (FRhL), MRC5, human embryonic kidney (HEK293), primary dog kidney, primary rabbit kidney, xenopus oocytes, or chick embryo cells.
  • DNA compounds differing in their nucleotide sequences can be used to encode a given polypeptide of the disclosure (e.g., an envelope and/or pre- membrane polypeptide).
  • the native DNA sequence encoding the polypeptides described herein are referenced merely to illustrate an embodiment of the disclosure, and the disclosure includes DNA compounds of any sequence that encode the amino acid sequences of the polypeptides utilized in the methods of the disclosure.
  • a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired immunogenicity.
  • the disclosure includes such polypeptides with different amino acid sequences than the specific proteins described herein so long as the modified or variant polypeptides have the desired immunogenicity of the reference polypeptide.
  • the polypeptides described herein e.g., envelope and/or pre-membrane polypeptides
  • the chimeric flavivirus vectors of the invention encode a structural protein from a first flavivirus with a low level of replication in a cell.
  • the first flavivirus with a low level of replication in a cell is a dengue virus.
  • the dengue virus may be selected from the group consisting of dengue type 1 (DEN1) virus, dengue type 2 (DEN2) virus, dengue type 3 (DEN3) virus, and dengue type 4 (DEN4) virus.
  • DEN1 dengue type 1
  • DEN2 dengue type 2
  • DEN3 dengue type 3
  • DEN4 dengue type 4
  • the present invention provides, in part, inactivated chimeric flaviviruses derived from chimeric flavivirus vectors comprising a sequence encoding an envelope protein and optionally a pre-membrane protein from a dengue virus for use in vaccines.
  • an inactivated, whole virion dengue vaccine provides a number of advantages over previous approaches, e.g., (a) the requirement of no more than two doses to achieve complete immunization (whereas three doses are required for the leading live vaccine); (b) no interference between non-replicating antigens in a tetravalent mixture; (c) 90-100% of subjects develop antibody to all four serotypes; and (d) since interference does not occur, the two doses of vaccine can be given at short intervals, e.g., 14, 21, or 28 days (whereas the leading live vaccine requires dosing spaced apart at approximately 3-6 months).
  • the first flavivirus with a low level of replication in a cell is a yellow fever virus.
  • the present invention provides, in part, inactivated chimeric flaviviruses derived from chimeric flavivirus vectors comprising an envelope protein and/or pre-membrane protein from a yellow fever virus for use in vaccines.
  • an inactivated, whole virion yellow fever vaccine provides a key advantage over previous approaches which utilize a live, attenuated vaccine.
  • live, attenuated yellow fever vaccines which can be associated with adverse events including potentially serious and fatal adverse neurotropic and viscerotropic events
  • the use of an inactivated, whole virion yellow fever vaccine mitigates the potential for serious adverse events associated with the currently available live, attenuated vaccines.
  • the chimeric flavivirus vectors of the invention comprise a backbone from a second flavivirus with a high level of replication in the cell.
  • the backbone from a second flavivirus with a high level of replication in the cell comprises a capsid protein.
  • the backbone from a second flavivirus with a high level of replication in the cell comprises one or more non- structural proteins.
  • the backbone from a second flavivirus with a high level of replication in the cell comprises all non- structural proteins.
  • the backbone from a second flavivirus with a high level of replication in the cell comprises the 3 ' and 5' noncoding termini.
  • the backbone from a second flavivirus with a high level of replication in the cell comprises a capsid protein, one or more non-structural proteins, and the 3' and 5 ' noncoding termini.
  • the backbone from a second flavivirus with a high level of replication in the cell comprises a capsid protein, all non-structural proteins, and the 3' and 5' noncoding termini.
  • the second flavivirus is a flavivirus capable of high level replication in a cell and replicates without causing extensive cytopathic effects (CPE) in the cell, e.g., without causing substantial degenerative changes in the cell.
  • the second flavivirus with a high level of replication in a cell can be a flavivirus capable of high level replication in a cell as well as allowing more than one harvest of cell culture fluid, e.g. , multiple harvest of flavivirus containing cell culture fluid.
  • the second flavivirus with a high level of replication in a cell can be a flavivirus capable of high level replication in a cell as well as causing a persistent and non-pathogenic infection, e.g.
  • infected cell culture allows infected cell culture to remain intact over a period of time ⁇ e.g., more than one day) and permit harvest of the cell culture supernatant fluid over a period of time ⁇ e.g., up to 4, 5, 6, or 7 days with daily harvest) to increase the volume of virus-containing cell culture fluid collected in every batch.
  • the second flavivirus is a flavivirus capable of high level replication in a cell and replicates without causing a significant elevation in the extracellular release of DNA, e.g., host DNA release into cell culture medium.
  • the second flavivirus is a flavivirus that does not cause an elevation, e.g., a measurable elevation in extracellular DNA release as compared to a mock infection when growing in a cell culture, and as measured at day 3 post-infection.
  • the second flavivirus is a flavivirus that does not cause an elevation in extracellular DNA release at more than 5 ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL, 30 ng/mL, or 100 ng/mL as compared to a mock infection when growing in a cell culture and measured at day 3 post-infection [0098]
  • the second flavivirus is a flavivirus selected from the Rio Bravo taxonomic group.
  • the flavivirus of the Rio Bravo taxonomic group may be selected from the group consisting of the Rio Bravo (sensu stricto) virus, the Montana Myotis Leukoencephalitis virus, the Dakar Bat virus, the Phnom Penh Bat virus, the Carey Island virus, and the Bukalasa Bat virus.
  • the flavivirus of the Rio Bravo taxonomic group is the Rio Bravo virus ⁇ sensu stricto) virus.
  • the phylogenetic relationship of the Rio Bravo taxonomic group of flaviviruses with the other members of the Flavivirus genus is illustrated in Figure 1.
  • the invention is based, in part, on the discovery that members of the Rio Bravo taxonomic group are capable of replicating to very high titers (> 10 8 PFUs/mL) in acceptable cell lines for manufacturing ⁇ e.g., Vera cells).
  • Members of the Rio Bravo taxonomic group of viruses are not transmitted by arthropod vectors and are instead maintained in nature by direct transmission between a specific order of vertebrates (Chiroptera bats).
  • the Rio Bravo virus has the advantage that they can be manipulated under Biological Safety Level 2 (BSL2) conditions, whereas the West Nile virus requires higher containment conditions (BSL3).
  • Rio Bravo virus is capable of replicating at high level in cells without causing extensive cytopathic effects in cells and/or elevation in the extracellular release of host DNA so that multiple harvest of cell culture fluid can be made during manufacturing batches.
  • the second flavivirus is a flavivirus selected from the Philippine S taxonomic group.
  • the flavivirus of the Kenya S taxonomic group may be selected from the group consisting of the Kenya S virus, the Banzi virus, and the Jugra virus.
  • the phylogenetic relationship of the Kenya S taxonomic group of flaviviruses with the other members of the Flavivirus genus is also illustrated in Figure 1. Similar to the Rio Bravo virus, the Philippine S taxonomic group of flaviviruses has the advantage that they can be manipulated under Biological Safety Level 2 (BSL2) conditions.
  • BSL2 Bio Safety Level 2
  • the second flavivirus is a flavivirus selected from the mosquito-associated flaviviruses.
  • the mosquito-associated flavivirus may be selected from the group consisting of the Kamiti River virus, the Culex flavivirus, the Aedes flavivirus, the Nakiwogo virus, the Quang Binh virus, and the Cell Fusing Agent virus. These mosquito-associated flaviviruses represent primitive insect viruses that are incapable of infecting mammalian cells.
  • viruses isolated from mosquitoes belonging to the genera Culex, Aedes, or Melanoconion mosquitoes include the Cell Fusing Agent virus (Stollar et al, 1975, Virology 64: 367-77), the Kamiti River virus (Crabtree et al, 2003, Arch Virol. 148: 1095-1118), the Culex flavivirus (Blivich et al, 2009, J Med Entomol.
  • the chimeric flavivirus vectors comprising a backbone from a mosquito-associated flaviviruses may be capable of replicating in insect cells, but not capable of replicating in human cells.
  • insect cell lines for use in the present invention include u4.4 cells, High FiveTM cells, Schneider's Drosophila cell line 2, Spodoptera frugiperda SF9 cells, Anopheles albimanus cells, Anopheles gambiae cells, Culex tarsalis cells, Phlebotomus papatasi cells, and C7-10 cells.
  • chimeric flaviviruses derived from chimeric flavivirus vectors comprising a backbone from a mosquito-associated flaviviruses may be used in a vaccine without prior inactivation. Further, as described herein, such chimeric flaviviruses derived from chimeric flavivirus vectors comprising a backbone from a mosquito-associated flaviviruses may be used in a vaccine without the addition of an adjuvant, because the viral RNA genome remains intact and acts as a self-adjuvant.
  • second flavivirus may be a flavivirus selected from the group consisting of the West Nile virus, the Rocio virus, the Ilheus virus, the Japanese encephalitis virus or the Murray Valley encephalitis virus.
  • the chimeric flavivirus vector encodes a chimeric flavivirus capable of replicating in mammalian cells to at least about 10 8 PFU/mL. In a further embodiment, the chimeric flavivirus vector encodes a chimeric flavivirus capable of replicating in mammalian cells to at least about 10 9 PFU/mL. [00105] As described above, in some embodiments, the chimeric flavivirus vectors of the invention comprise a backbone from a flavivirus with a high level of replication in the cell.
  • serial passages may be employed in cell culture to "adapt" the virus to grow to even higher titer. This step may result in specific mutations or deletions in the virus genome.
  • the adapted-mutated virus strain may be used to prepare an infectious clone, as described herein.
  • the adaptation mutations can be inserted by site-directed mutagenesis, resulting in a virus that grows optimally in the selected cell culture.
  • the vector virus strain After selection of the vector virus strain based on its growth characteristics, it is adapted by serial passage in selected cell type, and a stock of virus prepared by passage in the same cells. Another growth curve is performed to confirm that the virus yield is similar to that seen with the unadapted virus.
  • the R A of the adapted virus e.g., Rio Bravo virus
  • the R A of the adapted virus is extracted and reverse transcribed to the complementary DNA sequence using reverse transcriptase for sequencing to determine which mutations have occurred that are associated with adaptation to the cell substrate.
  • the backbone may be selected from a flavivirus that has been adapted to a cell substrate.
  • the backbone may be selected from a flavivirus that has been adapted to Vera, Madin Darby Canine kidney (MDCK), A549, fetal rhesus lung (FRhL), MRC5, human embryonic kidney (HEK293), primary dog kidney, primary rabbit kidney, xenopus oocytes, or chick embryo cells.
  • the backbone is selected from a flavivirus adapted to a cell substrate such that an increase in yield of the virus is at least about 2.5 -fold as compared to the flavivirus before adaptation.
  • the backbone may comprise at least one amino acid modification in a non-structural protein derived from a flavivirus with a high level of replication in the cell.
  • the non-structural protein is selected from the group consisting of NS1 , NS2A, NS2B, NS3, NS4A, NS4B, and NS5.
  • the backbone may comprise at least one amino acid modification in at least two non-structural proteins selected from the group consisting of NS1 , NS2A, NS2B, NS3, NS4A, NS4B, and NS5.
  • the non-structural protein is NS 1.
  • the NS1 protein may comprise a substitution at an amino acid position corresponding to the proline 315 residue of the Rio Bravo virus NS1 (SEQ ID NO: 1).
  • the proline 315 residue of the NS1 protein is replaced with a serine residue.
  • the non-structural protein is NS3.
  • the NS3 protein may comprise a substitution at an amino acid position corresponding to the isoleucine 555 residue of the Rio Bravo virus NS3 (SEQ ID NO: 2).
  • the isoleucine 555 residue of the NS3 protein is replaced with a threonine residue.
  • the chimeric flavivirus vector may comprise at least one nucleotide deletion in the 3' non-coding region (NCR).
  • the 3' non-coding region may comprise a nucleotide deletion at a nucleotide corresponding to the thymine at position 10692 of the Rio Bravo virus genome.
  • the chimeric flavivirus vector may comprise at least one nucleotide deletion in the 5' non-coding region (NCR).
  • the chimeric flavivirus vector may further comprise a signal sequence at the 3' end of the capsid gene.
  • the chimeric flavivirus vector may be within a plasmid comprising at least one cytomegalovirus (CMV) promoter, e.g., operably linked to the chimeric flavivirus vector.
  • CMV cytomegalovirus
  • the chimeric flavivirus vector may be within a plasmid expressing at least one hepatitis ⁇ virus ribozyme.
  • the chimeric flavivirus vector may be within a plasmid comprising at least one SV40 polyadenylation site.
  • the chimeric flavivirus vector may comprise one or more intron sequences.
  • the chimeric flavivirus vector may comprise an intron at the junction of the envelope (E) and the non- structural gene 1 (NS1).
  • the chimeric flavivirus vector may comprise an intron immediately after a nucleotide corresponding to position 9742 of the Rio Bravo virus genome.
  • the invention provides chimeric flaviviruses encoded by a chimeric flavivirus vector described herein.
  • the chimeric flavivirus vector comprises a sequence encoding a structural protein from a first flavivirus with a low level of replication in a cell and a backbone from a second flavivirus with a high level of replication in the cell.
  • the chimeric flavivirus vector encodes a chimeric flavivirus, wherein the chimeric flavivirus comprises a structural protein from a first flavivirus with a low level of replication in a cell and 1) a capsid protein from a second flavivirus with a high level of replication in the cell, 2) an R A genome comprising a sequence encoding one or more or all of the non-structural proteins, e.g., NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 from a second flavivirus with a high level of replication in the cell, or 3) an RNA genome with both 3' and 5' termini of the genome from a second flavivirus with a high level of replication in the cell, or a combination thereof.
  • the chimeric flavivirus comprises a structural protein from a first flavivirus with a low level of replication in a cell and 1) a capsid protein from a second flavivirus with a high level of replication
  • the invention provides methods of culturing one or more chimeric flavivirus vectors to produce chimeric flavivirus, e.g., growing chimeric flavivirus in cell cultures.
  • the chimeric flavivirus vector comprises a sequence encoding a structural protein from a first flavivirus with a low level of replication in a cell and a backbone from a second flavivirus with a high level of replication in the cell.
  • the chimeric flavivirus vector encodes a chimeric flavivirus, wherein the chimeric flavivirus comprises a structural protein from a first flavivirus with a low level of replication in a cell and 1) a capsid protein from a second flavivirus with a high level of replication in the cell, 2) an RNA genome comprising a sequence encoding one or more or all of the non-structural proteins, e.g., NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 from a second flavivirus with a high level of replication in the cell, or 3) an RNA genome with both 3' and 5' termini of the genome from a second flavivirus with a high level of replication in the cell, or a combination thereof.
  • the chimeric flavivirus comprises a structural protein from a first flavivirus with a low level of replication in a cell and 1) a capsid protein from a second flavivirus with a high level of replication
  • the one or more chimeric flavivirus vectors may be used to infect an appropriate cell line.
  • a cell culture strain for production is selected based on demonstrating maximum growth of both the second flavivirus with a high level of replication in the cell and the first flavivirus strain with a low level of replication in a cell which is donating a structural protein ⁇ e.g., an envelope and/or a pre -membrane protein).
  • the cells used for growth of the chimeric flavivirus can be one of a number of suitable cell cultures for vaccine development, including primary chick embryo cells, primary duck embryo cells, primary rabbit kidney, primary dog kidney, diploid continuous embryonic avian cell lines, mammalian diploid cells such as fetal rhesus lung (FRhL) or MRC5, or heteroploid cells such as Vero cells, PerC6, 293T, 293S, Madin Darby Canine Kidney (MDCK), human embryonic kidney (HEK293), xenopus oocytes, or A549 cells.
  • the cells are Vera cells.
  • chimeric flavivirus vectors comprising a backbone from a mosquito- associated flavivirus
  • these chimeric flavivirus vectors may be capable of replicating in insect cells, but not capable of replicating in human cells.
  • insect cell lines for use in culturing chimeric flavivirus vectors comprising a backbone from a mosquito- associated flavivirus include C6/36 Aedes albopictus mosquito cells, u4.4 cells, High FiveTM cells, Schneider's Drosophila cell line 2, Spodoptera frugiperda SF9 cells, Anopheles albimanus cells, Anopheles gambiae cells, Culex tarsalis cells, Phlebotomus papatasi cells, and CI- 10 cells.
  • the cell line may be cultured to produce a cell culture supernatant comprising the chimeric flavivirus, e.g., the virus is released into the cell culture supernatant from which it may be harvested.
  • a cell culture supernatant comprising the chimeric flavivirus
  • the virus is released into the cell culture supernatant from which it may be harvested.
  • growth of the chimeric flavivirus in cell culture is performed without the addition of bovine serum, porcine trypsin and other animal derived products which can be the source of adventitious viruses.
  • the cell culture may be grown in suspension, on microcarrier beads, or on the surface of tissue culture flasks, cell factories or roller bottles.
  • the chimeric flavivirus may be harvested from the cell culture supernatant.
  • the chimeric flavivirus vector encodes a chimeric flavivirus capable of replicating in mammalian cells to at least about 10 8 PFU/mL. In a further embodiment, the chimeric flavivirus vector encodes a chimeric flavivirus capable of replicating in mammalian cells to at least about 10 9 PFU/mL. In additional embodiments, the chimeric flavivirus vector encodes a chimeric flavivirus capable of replicating in insect cells, but not capable of replicating in human cells.
  • the invention provides an inactivated chimeric flavivirus encoded by a chimeric flavivirus vector described herein.
  • the chimeric flavivirus comprises a structural protein from a first flavivirus with a low level of replication in a cell, e.g., an envelope protein and optionally a membrane protein and 1) a capsid protein from a second flavivirus with a high level of replication in the cell, 2) an R A genome comprising a sequence encoding one or more or all of the non-structural proteins, e.g., NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 from a second flavivirus with a high level of replication in the cell, or 3) an RNA genome with both 3' and 5' termini of the genome from a second flavivirus with a high level of replication in the cell, or a combination thereof.
  • an inactivated chimeric flavivirus can be obtained by routine methods well known to the person skilled in the art. General methods for the inactivation of viral pathogens are described in US 2006/0270017, which is hereby incorporated by reference in its entirety. Downstream manufacturing steps used to purify and inactivate whole virion vaccines against other Flaviviruses ⁇ e.g., Japanese encephalitis, tick- borne encephalitis, and West Nile virus) have been described previously. See, e.g., WO/2006/122964. See also Srivastava et al, 2001, Vaccine 19: 4557-65.
  • the chimeric flavivirus encoded by a chimeric flavivirus vector may be inactivated with a method selected from the group consisting of chemical inactivation, high pressure inactivation, ultraviolet radiation, and gamma radiation.
  • the chimeric flavivirus is inactivated using chemical inactivation.
  • the method of chemical inactivation may comprise exposure of the flavivirus to one or more agents selected from the group consisting of ⁇ -propiolactone, formalin, aziridines, hydrogen peroxide, organic solvents, and ascorbic acid.
  • the chimeric flavivirus is inactivated may be inactivated by exposure to ⁇ - propiolactone.
  • the inactivated chimeric flavivirus may then be purified for use in various applications, including vaccine and other pharmaceutical compositions.
  • the method of inactivation further comprises a step of purifying the inactivated chimeric flavivirus in the sample to pharmaceutical purity and formulating the purified chimeric flavivirus into a pharmaceutical composition for use as a vaccine. Purification may be accomplished by any means known in the art, including, but not limited to, filtration or diafiltration, chromatography (e.g., size exclusion, ion exchange, immunoaffmity, and the like) or centrifugation. Alternatively, the chimeric flavivirus may be purified prior to inactivation by the methods of the invention.
  • the invention provides a vaccine comprising a chimeric flavivirus described herein.
  • the vaccine comprises an inactivated chimeric fiavivirus encoded by a chimeric flavivirus vector described herein.
  • the vaccine comprises an inactivated chimeric flavivirus, wherein the inactivated chimeric flavivirus is derived from a chimeric flavivirus comprising a structural protein, e.g.
  • the inactivated chimeric flavivirus is derived from a chimeric flavivirus comprising a structural protein, e.g., an envelope (E) and optionally a membrane protein from a first fiavivirus with a low level of replication in a cell, wherein the first flavivirus with a low level of replication in a cell is a dengue virus.
  • the dengue virus may be selected from the group consisting of dengue type 1 (DEN1) virus, dengue type 2 (DEN2) virus, dengue type 3 (DEN3) virus, and dengue type 4 (DEN4) virus.
  • the inactivated chimeric flavivirus is derived from a chimeric flavivirus comprising a structural protein, e.g., an envelope (E) and optionally a membrane protein from a first flavivirus with a low level of replication in a cell, wherein the first flavivirus with a low level of replication in a cell is a yellow fever virus.
  • the inactivated chimeric flavivirus is derived from a chimeric flavivirus comprising 1) a capsid protein from a second flavivirus with a high level of replication in the cell, 2) an RNA genome comprising a sequence encoding one or more or all of the non-structural proteins, e.g., NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 from a second flavivirus with a high level of replication in the cell, or 3) an RNA genome with both 3' and 5' termini of the genome from a second flavivirus with a high level of replication in the cell, or a combination thereof, wherein the second flavivirus is a flavivirus capable of high level replication in a cell and replicates without causing extensive cytopathic effects (CPE) in the cell, e.g., without causing substantial degenerative changes in the cell.
  • CPE cytopathic effects
  • the second flavivirus with a high level of replication in a cell can be a flavivirus capable of high level replication in a cell as well as allowing more than one harvest of cell culture fluid, e.g., multiple harvest of flavivirus containing cell culture fluid.
  • the second flavivirus with a high level of replication in a cell can be a flavivirus capable of high level replication in a cell as well as causing a persistent and non-pathogenic infection, e.g., allow infected cell culture to remain intact over a period of time ⁇ e.g., more than one day) and permit harvest of the cell culture supernatant fluid over a period of time ⁇ e.g., up to 4, 5, 6, or 7 days with daily harvest) to increase the volume of virus-containing cell culture fluid collected in every batch.
  • a persistent and non-pathogenic infection e.g., allow infected cell culture to remain intact over a period of time ⁇ e.g., more than one day
  • harvest of the cell culture supernatant fluid e.g., up to 4, 5, 6, or 7 days with daily harvest
  • the inactivated chimeric flavivirus is derived from a chimeric flavivirus comprising 1) a capsid protein from a second flavivirus with a high level of replication in the cell, 2) an RNA genome comprising a sequence encoding one or more or all of the non-structural proteins, e.g., NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 from a second flavivirus with a high level of replication in the cell, or 3) an RNA genome with both 3' and 5' termini of the genome from a second flavivirus with a high level of replication in the cell, or a combination thereof, wherein the second flavivirus is a flavivirus capable of high level replication in a cell and replicates without causing a significant elevation in the extracellular release of DNA, e.g., host DNA release into cell culture medium.
  • the second flavivirus is a flavivirus that does not cause an elevation, e.g., a measurable elevation in extracellular DNA release as compared to a mock infection when growing in a cell culture, and as measured at day 3 post-infection.
  • the second flavivirus is a flavivirus that does not cause an elevation in extracellular DNA release at more than 5 ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL, 30 ng/mL, or 100 ng/mL as compared to a mock infection when growing in a cell culture and measured at day 3 post-infection
  • the inactivated chimeric flavivirus is derived from a chimeric flavivirus comprising 1) a capsid protein from a second flavivirus with a high level of replication in the cell, 2) an RNA genome comprising a sequence encoding one or more or all of the non-structural proteins, e.g., NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 from a second flavivirus with a high level of replication in the cell, or 3) an RNA genome with both 3' and 5' termini of the genome from a second flavivirus with a high level of replication in the cell, or a combination thereof, wherein the second flavivirus with a high level of replication in the cell is a flavivirus selected from the Rio Bravo taxonomic group.
  • the flavivirus of the Rio Bravo taxonomic group may be selected from the group consisting of the Rio Bravo (sensu stricto) virus, the Montana Myotis Leukoencephalitis virus, the Dakar Bat virus, the Phnom Penh Bat virus, the Carey Island virus, and the Bukalasa Bat virus.
  • the inactivated chimeric flavivirus is derived from a chimeric flavivirus comprising 1) a capsid protein from a second flavivirus with a high level of replication in the cell, 2) an RNA genome comprising a sequence encoding one or more or all of the non-structural proteins, e.g., NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 from a second flavivirus with a high level of replication in the cell, or 3) an RNA genome with both 3 ' and 5 ' termini of the genome from a second flavivirus with a high level of replication in the cell, or a combination thereof, wherein the second flavivirus with a high level of replication in the cell is a flavivirus selected from the Kenya S taxonomic group.
  • the flavivirus of the Kenya S taxonomic group may be selected from the group consisting of the Kenya S virus, the Banzi virus, and the
  • the inactivated chimeric flavivirus is derived from a chimeric flavivirus comprising 1) a capsid protein from a second flavivirus with a high level of replication in the cell, 2) an RNA genome comprising a sequence encoding one or more or all of the non-structural proteins, e.g., NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 from a second flavivirus with a high level of replication in the cell, or 3) an RNA genome with both 3 ' and 5 ' termini of the genome from a second flavivirus with a high level of replication in the cell, or a combination thereof, wherein the second flavivirus with a high level of replication in the cell is a flavivirus selected from the mosquito-associated flaviviruses.
  • the mosquito-associated flavivirus may be selected from the group consisting of the Kamiti River virus, the Culex flavivirus, the Aedes flavivirus, the Nakiwogo virus, the Quang Binh virus, and the Cell Fusing Agent virus.
  • the inactivated chimeric flavivirus is derived from a chimeric flavivirus comprising 1) a capsid protein from a second flavivirus with a high level of replication in the cell, 2) an RNA genome comprising a sequence encoding one or more or all of the non-structural proteins, e.g., NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 from a second flavivirus with a high level of replication in the cell, or 3) an RNA genome with both 3' and 5' termini of the genome from a second flavivirus with a high level of replication in the cell, or a combination thereof, wherein the second flavivirus with a high level of replication in the cell may be a flavivirus selected from the group consisting of the West Nile virus, the Rocio virus, the Ilheus virus, the Japanese encephalitis virus or the Murray Valley encephalitis virus.
  • the vaccine comprises a live, chimeric flavivirus, wherein the live chimeric flavivirus comprises a structural protein from a first flavivirus with a low level of replication in a cell and 1) a capsid protein from a second flavivirus with a high level of replication in the cell, 2) an RNA genome comprising a sequence encoding one or more or all of the non-structural proteins, e.g., NSl, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 from a second flavivirus with a high level of replication in the cell, or 3) an RNA genome with both 3 ' and 5 ' termini of the genome from a second flavivirus with a high level of replication in the cell, or a combination thereof.
  • the live chimeric flavivirus comprises a structural protein from a first flavivirus with a low level of replication in a cell and 1) a capsid protein from a second flavivirus with a high level of replication
  • the live, chimeric flavivirus comprises a structural protein from a first flavivirus with a low level of replication in a cell and 1) a capsid protein from a mosquito-associated flavivirus, 2) an RNA genome comprising a sequence encoding one or more or all of the non-structural proteins, e.g., NSl, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 from a mosquito-associated flavivirus, or 3) an RNA genome with both 3' and 5' termini of the genome from a mosquito-associated flavivirus, or a combination thereof.
  • the mosquito-associated flavivirus may be selected from the group consisting of the Kamiti River virus, the Culex flavivirus, the Aedes flavivirus, the Nakiwogo virus, the Quang Binh virus, and the Cell Fusing Agent virus.
  • the vaccine comprising a live, chimeric flavivirus may be administered to a subject in need thereof in the absence of adjuvant.
  • the invention provides a vaccine comprising a chimeric flavivirus vector described herein.
  • the invention provides a DNA vaccine comprising a chimeric flavivirus vector described herein.
  • the invention provides a DNA vaccine comprising a chimeric flavivirus vector described herein and suitable for delivery by gene gun or any other DNA vaccine delivery methods.
  • the term "vaccine” refers to a composition that comprises one or more chimeric flavivirus vectors and/or chimeric flaviviruses of the invention.
  • the chimeric flavivirus of the vaccine is an inactivated chimeric flavivirus.
  • the chimeric flavivirus of the vaccine is a live, chimeric flavivirus.
  • the vaccine may comprise a mixture of a first, second, third, and/or fourth chimeric flavivirus vectors wherein the envelope protein in the first, second, third, and/or fourth chimeric flavivirus vector is derived from a dengue type 1 (DEN1) virus, dengue type 2 (DEN2) virus, dengue type 3 (DEN3) virus, and dengue type 4 (DEN 4) virus, respectively.
  • DEN1 dengue type 1
  • DEV3 dengue type 3
  • DEN4 dengue type 4
  • the vaccine may comprise a mixture of a first, second, third, and/or fourth chimeric flaviviruses encoded by a first, second, third, and/or fourth chimeric flavivirus vector, wherein the envelope protein in the first, second, third, and/or fourth is derived from a dengue type 1 (DEN1) virus, dengue type 2 (DEN2) virus, dengue type 3 (DEN3) virus, and dengue type 4 (DEN 4) virus, respectively.
  • DEN1 dengue type 1
  • DEN2 dengue type 2
  • DEN3 dengue type 3
  • DEN4 dengue type 4 virus
  • one or more of the chimeric flaviviruses of said mixture has been inactivated.
  • all chimeric flaviviruses of said mixture have been inactivated.
  • one or more of the chimeric flaviviruses of said mixture is a live, chimeric flavivirus.
  • all chimeric flaviviruses of said mixture are live, chimeric flaviviruses.
  • the vaccine further comprises an adjuvant.
  • adjuvants include, but are not limited to, salts, such as calcium phosphate, aluminum phosphate, calcium hydroxide and aluminum hydroxide; natural polymers such as algal glucans (e.g., beta glucans), chitosan or crystallized inulin; synthetic polymers such as poly- lactides, poly-glycolides, poly lacitide-co-glycolides or methylacrylate polymers; oil-in-water emulsions such as MF59; water-in-oil emulsions, micelle-forming cationic or non-ionic block copolymers or surfactants such as Pluronics, L121 , 122 or 123, Tween 80, or NP-40; fatty acid, lipid (e.g.
  • lipid A or monophosphoryl lipid A) or lipid and protein based vesicles such as liposomes, proteoliposomes, ISCOM, ISCOMATRIX, and cochleate structures; surfactant stabilized emulsions composed of synthetic or natural oils and aqueous solutions, immunostimulatory oligonucleotides (e.g. , CpGs), and poly I:C.
  • a vaccine of the invention upon administration to a subject, is capable of stimulating an immune response (e.g. , a humoral immune response, cellular immune response, or both) in the subject.
  • the immune response includes a measurable response (e.g. , a measurable humoral or cellular immune response, or combination thereof) to an epitope encoded by a structural protein (e.g. , a dengue virus or yellow fever virus envelope and/or a membrane protein) inserted or integrated into a chimeric flavivirus of the vaccine.
  • a vaccine of the invention is capable of providing protection against dengue.
  • a vaccine of the invention is capable of providing protection against yellow fever.
  • the vaccine is capable of stimulating an immune response against one or more antigens (e.g. , encoded by a dengue virus or yellow fever virus structural protein) such that, upon later encountering such an antigen, the subject receiving the vaccine has an immune response that is stronger than it would have been if the vaccine had not been administered previously.
  • a vaccine of the invention is capable of ameliorating a dengue virus infection and/or reducing at least one symptom of dengue virus infection.
  • a vaccine of the invention is capable of ameliorating a yellow fever virus infection and/or reducing at least one symptom of yellow fever virus infection.
  • the vaccine of the invention induces a therapeutic immune response against one or more antigens (e.g., encoded by a dengue virus or yellow fever virus structural protein) such that symptoms and/or complications of an infection in a subject suffering from such an infection.
  • one or more antigens e.g., encoded by a dengue virus or yellow fever virus structural protein
  • the chimeric flavivirus vectors and/or chimeric flaviviruses used for the vaccines can be prepared and formulated for administration to a mammal in accordance with techniques well known in the art.
  • Formulations for oral administration can consist of capsules or tablets containing a predetermined amount of a chimeric flavivirus of the invention; liquid solutions, such as an effective amount of the pharmaceutical dissolved in ingestible diluents, such as water, saline, orange juice, and the like; suspensions in an appropriate liquid; and suitable emulsions.
  • the chimeric flavivirus vectors and/or chimeric flaviviruses of the invention can, for example, be formulated as enteric coated capsules for oral administration, as previously described, in order to bypass the upper respiratory tract and allow viral replication in the gut. See, e.g., Tacket et al., 1992, Vaccine 10: 673-6; Horwitz, in Fields et al., eds., 1996, Fields Virology, third edition, Vol 2: 2149-71; Takafuji et al, 1979, J. Infec. Dis. 140: 48-53; and Top et al., 1971, J. Infec. Dis. 124: 155-60.
  • the chimeric flaviviruses can be formulated in conventional solutions, such as sterile saline, and can incorporate one or more pharmaceutically acceptable carriers or excipients.
  • the pharmaceutical composition can further comprise other active agents.
  • formulations of the invention comprise a buffered solution comprising one or more chimeric flaviviruses of the invention in a pharmaceutically acceptable carrier.
  • carriers can be used, such as buffered saline, water and the like.
  • Such solutions are generally sterile and free of undesirable matter.
  • These compositions can be sterilized by conventional, well known sterilization techniques, or can be sterile filtered.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • Pharmaceutically acceptable carriers can contain a physiologically acceptable compound that acts, e.g., to stabilize the composition or to increase or decrease the absorption of the virus and/or pharmaceutical composition.
  • Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of any co-administered agents, or excipient, or other stabilizers and/or buffers.
  • Detergents can also be used to stabilize the composition or to increase or decrease absorption. Detergents may be used to 'split' the virus by disrupting the lipid-containing envelope of the virion.
  • a pharmaceutically acceptable carrier including a physiologically acceptable compound depends, e.g., on the route of administration of the adenoviral preparation and on the particular physio-chemical characteristics of any coadministered agent.
  • the chimeric flavivirus vectors and/or chimeric flaviviruses of the invention can also be administered in a lipid formulation, more particularly either complexed with liposomes or to lipid/nucleic acid complexes or encapsulated in liposomes.
  • the chimeric flavivirus vectors and/or chimeric flaviviruses of the current invention alone or in combination with other suitable components, can also be made into aerosol formulations to be administered via inhalation.
  • the vaccines can also be formulated for administration via the nasal passages.
  • Formulations suitable for nasal administration wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 10 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • Suitable formulations wherein the carrier is a liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebulizer include aqueous or oily solutions of the active ingredient.
  • the chimeric flaviviruses of the invention can be formulated as suppositories, for example, for rectal or vaginal administration.
  • DNA vaccines e.g., vaccines comprising a chimeric flavivirus vector of the invention
  • these DNA vaccines can be delivered by different routes of administration.
  • the DNA vaccines e.g., vaccines comprising a chimeric flavivirus vector described herein
  • the DNA vaccine may be administered by routes including, but not limited to, inhalation, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, subcutaneous injection, application to mucosal surfaces (e.g., application of DNA drops to the nares or trachea), intraocular administration, or particle bombardment of the epidermis using a gene gun.
  • DNA vaccines can be injected in saline solutions into muscle or skin using a syringe and needle. See, e.g., US 2002/0025939. DNA vaccines can also be administered by coating the nucleic acid onto microscopic gold beads and then using a gene gun to deliver the beads into cells. The saline injections deliver the nucleic acid into extracellular spaces, whereas gene guns deliver nucleic acid coated gold beads directly into cells.
  • Vaccines comprising a chimeric flavivirus vectors and/or chimeric flavivirus of the invention can have a unit dosage comprising between about 5 ⁇ g to about 100 ⁇ g (e.g., about 5 ⁇ g to about 15 ⁇ g, about 15 ⁇ g to about 25 ⁇ g, about 25 ⁇ g to about 35 ⁇ g, about 35 ⁇ g to about 45 ⁇ g, about 45 ⁇ g to about 55 ⁇ g, about 55 ⁇ g to about 65 ⁇ g, about 65 ⁇ g to about 75 ⁇ g, about 75 ⁇ g to about 85 ⁇ g, about 85 ⁇ g to about 95 ⁇ g, and about 95 ⁇ g to about 100 ⁇ g) of the chimeric flavivirus in a single dose.
  • a unit dosage comprising between about 5 ⁇ g to about 100 ⁇ g (e.g., about 5 ⁇ g to about 15 ⁇ g, about 15 ⁇ g to about 25 ⁇ g, about 25 ⁇ g to about 35 ⁇ g, about 35
  • the dosages can vary based on the route of administration.
  • vaccines formulated for sublingual or intranasal administration may contain a lower dosage of chimeric flavivirus per single dose than vaccines formulated for alternative routes of administration, e.g., the oral routes of administration.
  • alternative routes of administration e.g., the oral routes of administration.
  • One of skill in the art can determine the appropriate dosage for a particular patient depending on the type of infection, and the route of administration to be used without undue experimentation.
  • the invention provides a method for inducing an immune response in a subject, wherein the method comprises administration of a chimeric flavivirus vectors described herein and/or chimeric flavivirus encoded by a chimeric flavivirus vector described herein.
  • the administration includes a single dose administration.
  • the administration includes no more than two, three, or four doses of administration.
  • the chimeric flavivirus is an inactivated chimeric flavivirus.
  • the chimeric flavivirus comprises a structural protein, e.g., an envelope (E) and optionally a membrane protein from a first flavivirus with a low level of replication in a cell and 1) a capsid protein from a second flavivirus with a high level of replication in the cell, 2) an RNA genome comprising a sequence encoding one or more or all of the non-structural proteins, e.g., NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 from a second flavivirus with a high level of replication in the cell, or 3) an RNA genome with both 3' and 5' termini of the genome from a second flavivirus with a high level of replication in the cell, or a combination thereof.
  • the inactivated chimeric flavivirus is administered in conjunction with an adjuvant.
  • the invention provides a method for inducing an immune response in a subject, wherein the method comprises administration of a live, chimeric flavivirus encoded by a chimeric flavivirus vector described herein.
  • the live, chimeric flavivirus comprises a structural protein from a first flavivirus with a low level of replication in a cell and 1) a capsid protein from a mosquito-associated flavivirus, 2) an RNA genome comprising a sequence encoding one or more or all of the nonstructural proteins, e.g., NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 from a mosquito- associated flavivirus, or 3) an RNA genome with both 3 ' and 5' termini of the genome from a mosquito-associated flavivirus, or a combination thereof.
  • the mosquito-associated flavivirus may be selected from the group consisting of the Kamiti River virus, the Culex flavivirus, the Aedes flavivirus, the Nakiwogo virus, the Quang Binh virus, and the Cell Fusing Agent virus.
  • the vaccine comprising a live, chimeric flavivirus may be administered to a subject in need thereof in the absence of adjuvant.
  • the invention provides methods of inducing an immune response to dengue virus in a subject comprising administering to the subject a vaccine of the invention.
  • the vaccine may comprise a mixture of a first, second, third, and/or fourth chimeric flavivirus vectors wherein the envelope protein in the first, second, third, and/or fourth is derived from a dengue type 1 (DEN1) virus, dengue type 2 (DEN2) virus, dengue type 3 (DEN3) virus, and dengue type 4 (DEN 4) virus, respectively.
  • the vaccine may comprise a mixture of chimeric flaviviruses encoded by a first, second, third, and/or fourth chimeric flavivirus vector, wherein the envelope protein in the first, second, third, and/or fourth is derived from a dengue type 1 (DEN1) virus, dengue type 2 (DEN2) virus, dengue type 3 (DEN3) virus, and dengue type 4 (DEN 4) virus, respectively.
  • DEN1 dengue type 1
  • DEN2 dengue type 2
  • DEN3 dengue type 3
  • DEN4 dengue type 4
  • one or more of the chimeric flaviviruses of said mixture has been inactivated.
  • all chimeric flaviviruses of said mixture have been inactivated.
  • one or more of the chimeric flaviviruses of said mixture is a live, chimeric flavivirus.
  • all chimeric flaviviruses of said mixture are live, chimeric flaviviruses.
  • the invention provides methods of inducing an immune response to yellow fever virus in a subject comprising administering to the subject a vaccine of the invention.
  • the vaccine may comprise a chimeric flavivirus, wherein the chimeric flavivirus is derived from a chimeric flavivirus vector comprising a sequence encoding a structural protein from a yellow fever virus and a backbone from a second flavivirus with a high level of replication in the cell.
  • the vaccine may comprise a chimeric flavivirus vector, comprising a sequence encoding a structural protein from a yellow fever virus and a backbone from a second flavivirus with a high level of replication in the cell.
  • the invention provides a method of vaccinating a subject against an infectious pathogen comprising administering a sufficient amount of a vaccine of the invention to a subject at risk for being infected by an infectious pathogen.
  • the administration includes a single dose administration. In another embodiment, the administration includes no more than two, three, or four doses of administration.
  • the vaccine comprises the chimeric flavivirus vector of the invention. In some other embodiment, the vaccine comprises an inactivated chimeric flavivirus.
  • the chimeric flavivirus comprises a structural protein, e.g., an envelope (E) and optionally a membrane protein from a first flavivirus with a low level of replication in a cell and 1) a capsid protein from a second flavivirus with a high level of replication in the cell, 2) an RNA genome comprising a sequence encoding one or more or all of the non-structural proteins, e.g., NSl, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 from a second flavivirus with a high level of replication in the cell, or 3) an RNA genome with both 3 ' and 5 ' termini of the genome from a second flavivirus with a high level of replication in the cell, or a combination thereof
  • a structural protein e.g., an envelope (E) and optionally a membrane protein from a first flavivirus with a low level of replication in a cell and 1) a capsid protein from a second
  • the inactivated chimeric flavivirus is administered in conjunction with an adjuvant.
  • the vaccine comprises a live, chimeric flavivirus.
  • the vaccine comprises a live, chimeric flavivirus, wherein the live chimeric flavivirus comprises a structural protein from a first flavivirus with a low level of replication in a cell and 1) a capsid protein from a mosquito- associated flavivirus, 2) an RNA genome comprising a sequence encoding one or more or all of the non-structural proteins, e.g., NSl, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 from a mosquito-associated flavivirus, or 3) an RNA genome with both 3' and 5' termini of the genome from a mosquito-associated flavivirus, or a combination thereof.
  • the mosquito-associated flavivirus may be selected from the group consisting of the Kamiti River virus, the Culex flavivirus, the Aedes flavivirus, the Nakiwogo virus, the Quang Binh virus, and the Cell Fusing Agent virus.
  • the vaccine comprising a live chimeric flavivirus may be administered to a subject in need thereof in the absence of adjuvant.
  • the invention provides a method of vaccinating a subject against a dengue virus infection comprising administering to the subject a vaccine of the invention.
  • the vaccine may comprise a mixture of chimeric flaviviruses encoded by a first, second, third, and/or fourth chimeric flavivirus vector, wherein the envelope protein in the first, second, third, and/or fourth is derived from a dengue type 1 (DEN1) virus, dengue type 2 (DEN2) virus, dengue type 3 (DEN3) virus, and dengue type 4 (DEN 4) virus, respectively.
  • DEN1 dengue type 1
  • DEN2 dengue type 2
  • DEN3 dengue type 3
  • DEN4 dengue type 4
  • one or more of the chimeric flaviviruses of said mixture has been inactivated.
  • all chimeric flaviviruses of said mixture have been inactivated.
  • one or more of the chimeric flaviviruses of said mixture is a live chimeric flavivirus.
  • all chimeric flaviviruses of said mixture are live chimeric flaviviruses.
  • the vaccine may comprise a chimeric flavivirus, wherein the chimeric flavivirus is derived from a chimeric flavivirus vector comprising a sequence encoding a structural protein from a yellow fever virus and a backbone from a second flavivirus with a high level of replication in the cell.
  • the subject has an infection induced by the infectious pathogen (e.g., dengue virus or yellow fever virus).
  • the infectious pathogen e.g., dengue virus or yellow fever virus.
  • the present invention provides a method of inducing a therapeutic immune response in a subject experiencing an infection induced by an infectious pathogen (e.g., dengue virus or yellow fever virus).
  • an infectious pathogen e.g., dengue virus or yellow fever virus.
  • one or more symptoms or complications of the infection e.g., dengue virus infection or yellow fever virus infection
  • the vaccines of the invention can be used to vaccinate human or veterinary subjects.
  • the vaccines of the invention can be administered alone, or can be co-administered or sequentially administered with other immunological, antigenic, vaccine, or therapeutic compositions.
  • Such compositions can include other agents to potentiate or broaden the immune response, e.g., IL-2 or other cytokines which can be administered at specified intervals of time, or continuously administered (see, e.g., Smith et ah, 1997, N Engl J Med 336(17): 1260-1; and Smith et al, 1997, Cancer J Sci Am. 3 Suppl 1 : S137-40).
  • the vaccines of the invention can also be administered in conjunction with other vaccines, vectors, or viruses.
  • a chimeric flavivirus of the invention can be administered either before or after administration of another vaccine, e.g., another vaccine to an unrelated agents including without any limitation hepatitis A, hepatitis B, or typhoid vaccines or a combination thereof.
  • another vaccine e.g., another vaccine to an unrelated agents including without any limitation hepatitis A, hepatitis B, or typhoid vaccines or a combination thereof.
  • the chimeric flavivirus vectors and/or chimeric flavivirus formulations can be delivered by different routes of administration, e.g., systemically, regionally, or locally.
  • Regional administration refers to administration into a specific anatomical space, such as intraperitoneal, intrathecal, subdural, or to a specific organ, and the like.
  • Local administration refers to administration of a composition into a limited, or circumscribed, anatomic space such as subcutaneous injections, intramuscular injections, intradermal injections, or by application to the epidermis.
  • local administration or regional administration can also result in entry of the viral preparation into the circulatory system.
  • Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular or subcutaneous routes.
  • Other routes include oral administration, including administration to the oral mucosa (e.g., tonsils), intranasal, sublingual.
  • administration can also be performed via inhalation.
  • Aerosol formulations can, for example, be placed into pressurized, pharmaceutically acceptable propellants, such as dichlorodifluoro-methane, nitrogen and the like. They can also be formulated as pharmaceuticals for non-pressurized preparations such as in a nebulizer or an atomizer.
  • such administration is in an aqueous pharmacologically acceptable buffer as described above.
  • the vaccines of the invention can be administered in a variety of unit dosage forms, depending upon the intended use, e.g., prophylactic vaccine or therapeutic regimen, and the route of administration. With regard to therapeutic use, the particular condition or disease and the general medical condition of each patient will influence the dosing regimen.
  • the concentration of chimeric flavivirus in the pharmaceutically acceptable excipient can be, e.g., from about 5 ⁇ g to about 100 ⁇ g of chimeric flavivirus per dose, between about 15 ⁇ g to about 85 ⁇ g of chimeric flavivirus per dose, between about 25 ⁇ g to about 75 ⁇ g of chimeric flavivirus per dose, between about 35 ⁇ g to about 65 ⁇ g of chimeric flavivirus per dose, or between about 45 ⁇ g to about 55 ⁇ g of chimeric flavivirus per dose.
  • a therapeutically effective dose of a vaccine is an amount of chimeric flavivirus that will stimulate an immune response to the structural protein(s) encoded by the dengue virus or yellow fever virus nucleic acid included in the chimeric flavivirus vector.
  • the dosage schedule i.e., the dosing regimen, will depend upon a variety of factors, e.g. , the general state of the patient's health, physical status, age and the like. The state of the art allows the clinician to determine the dosage regimen for each individual patient.
  • Single or multiple administrations of the chimeric flavivirus vectors and/or chimeric flavivirus formulations can be administered as prophylactic vaccines.
  • multiple doses ⁇ e.g., two or more, three or more, four or more, or five or more doses) are administered to a subject to induce or boost a protective immune response.
  • the two or more doses can be separated by periodic intervals, for instance, one week, two week, three week, one month, two month, three month, or six month intervals.
  • only two doses of the vaccine are required and may be administered at short intervals (e.g. , two weeks apart, three weeks apart, or four weeks apart).
  • kits that contain the chimeric flavivirus vectors, chimeric flaviviruses, or vaccines of the invention.
  • the kits can, for example, also contain cells for growing the chimeric flaviviruses of the invention.
  • the kits can also include instructional material teaching methodologies for generating chimeric flaviviruses using the kits and, for vaccines, can include instruction for indication of dosages, routes and methods of administration and the like.
  • kits include containers suitable for transport and/or store flavivirus vectors, chimeric flaviviruses, or vaccines of the invention.
  • BSL2 viruses were selected by virtue of high growth potential belonging to a cluster of three phylogenetically related mosquito-borne viruses in the Edge Hill subgroup [Uganda S (UGS), Banzi (BAN), and Jugra (JUG)] (Grard et al, 2010, J Gen Virol 91 : 87-94).
  • a fourth BSL2 virus [Rio Bravo virus (RBV)] belongs to a distinct and different grouping of Flaviviruses having no known arthropod vector.
  • West Nile (WN) virus (BSL3) which is known to grow to high titer (9 logio PFU/mL) was selected as a comparator.
  • RBV achieved high yields (>8 log 10 PFU/mL) without causing cytopathic effects (CPE).
  • CPE cytopathic effects
  • typical of most flaviviruses, WN, UGS, BAN and JUG viruses caused extensive CPE beginning within a few hours of peak virus yields, and as the cells ceased macromolecular synthesis and underwent lysis or apoptosis, virus titers in the supernatant medium fell off rapidly (Figure 2A).
  • flaviviruses are released as mature, infectious virions into the cell culture medium; since they are unstable and lose infectivity rapidly outside of the cell at temperatures used for growth (37° C), the virus must be harvested at the peak titer, a critical, single point in time (within a period of 6-8 hr) which occurs when early CPE is evident.
  • RBV did little damage to cells, while still producing a high titer of virus ( Figure 2B).
  • replication of RBV continued for up to 7 days with continued release of high titers of virus.
  • the specific Rio Bravo virus used as the vector for constructing a chimeric virus may be one of the extant strains, including the Burns Bat strain (Texas, 1954), M64 (derived from the Burns bat strain), TVRL 126865) (Trinidad, 1973) or RiMAR (GenBank Accession AF 144692).
  • the specific strain used to determine the potential of Rio Bravo virus as a vector was the M64 strain.
  • M64 is considered the "prototype" strain of Rio Bravo virus (Catalogue of Arthropod-Borne Viruses, op cit).
  • This example illustrates how a Rio Bravo virus strain is adapted to increase the yield (titer) of virus in cell cultures.
  • the stock culture of Rio Bravo virus was passed by infecting duplicate monolayer culture of Vero cells in 25 cm 2 flasks at MOI 0.01. Blind passages were subsequently made of virus harvested in the early phase of growth cycle. At each passage, cell culture medium was removed from the flask, 0.1 mL of the virus to be passed is added to the flask and allowed to adsorb for 1 hr at 37°C, and the culture is washed to remove the inoculum, after which fresh medium is added. At each passage, 3 aliquots of each virus were frozen for titration.
  • nucleotide present on PI and on the right nucleotide present on P10 the
  • the Rio Bravo virus contained two distinct plaque populations, a small plaque virus (1 mm) and a large plaque population (5-6 mm).
  • the two plaque populations were seen at all passage levels (PI through P10).
  • the two plaque populations were separated by picking plaques in uncrowded cultures (two rounds) followed by a round of terminal dilution cloning.
  • the small and large plaque populations were then amplified by passage in monolayer cultures of Vera cells in serum free medium.
  • the small plaque population as a pure culture, has a slightly larger plaque size (2-3 mm) compared to the original small plaque (1 mm), whereas the large plaque population did not change (5-6 mm).
  • nucleotide present on PI and on the right nucleotide present on P10 the
  • NS3 nonstructural gene 3
  • RTPase RNA triphosphatase
  • the virus was used to infect different mammalian cell lines acceptable for manufacturing human vaccines. These included Vero (WHO 10-87), fetal rhesus lung (FRhL), A549, and Madin Darby Canine Kidney (MDCK) cells, all infected at MOI 0.01 PFU/cell. Vero cells produced the highest peak yield of virus as shown in Table 5.
  • the Rio Bravo virus-specific pre-membrane (prM) and envelope (E) genes within the full length infectious clone (FLIC) are replaced by the prM-E sequences of a selected dengue virus serotype using standard molecular cloning (including fusion PCR) techniques.
  • Rescue of the chimeric RB-DEN virus is obtained by transfection by electroporation or lipofectamine into Vera 10-87 cells and progeny virus is harvested by collection of the supernatant. Suitable passages are made to produce master and working virus seeds.
  • a full-length infectious clone of RBV is constructed.
  • the complete RBV genomic sequence is propagated in one plasmid under the expression of the cytomegalovirus (CMV) promoter at the 5 ' end.
  • CMV cytomegalovirus
  • a unique feature of the infectious clone is the insertion of the hepatitis ⁇ virus ribozyme (HDVr) immediately after the last nucleotide of Rio Bravo Virus cDNA sequence to ensure production of Rio Bravo Virus RNAs with the precise 3'- terminus, which is beneficial for more efficient RNA replication.
  • a SV40 polyadenylation site can be inserted downstream of the HDVr to ensure complete termination of transcription (Figure 7A).
  • Plasmid 2 contains a 300 nucleotide-long intron inserted by fusion PCR at the junction of the envelope (E) and non- structural gene 1 (NS1) to ensure stability of the resultant full-length cDNA template ( Figure 7B).
  • Plasmid 5 contains the hepatitis ⁇ virus ribozyme (HDVr) immediately after the last nucleotide of Rio Bravo Virus cDNA sequence, a SV40 polyadenylation site downstream of the HDVr, as well as a second intron inserted at nucleotide 9742 to increase stability of the construct, which is generated by fusion PCR ( Figure 7C).
  • the full-length cDNA template is generated sequentially by ligation with appropriate restriction fragments derived from these plasmids.
  • the RBV prME sequences are replaced by the corresponding DEN2 prME sequences that are produced by RT-PCR amplification of the specific virus genome, followed by fusion PCR to generate an amplicon incorporating the complete DEN2 prME sequence as well as RBV sequences spanning the BspEI and Sacl sites ( Figure 7D).
  • the chimeric RBV-DEN2 cDNA sequence is generated by digestion of the RBV cDNA and RB- DEN2 amplicon with the BspEI and Sacl restriction enzymes and ligation into the plasmid containing the RBV BspEI/SacI digested cDNA ( Figure 7E).
  • the resulting virus is sequenced to confirm the expected genome.
  • the signal sequence for furin proteolysis which represents the junction at the 5' end of the DEN2 donor prM gene is critical to successful generation of a viable chimera. It is thus ensured that the RBV vector signal sequence is retained in the recombinant infectious clone.
  • the recombinant infectious clone may be transfected by lipofection or electroporation into Vera 10-87 cells. Positive-sense RNA is then produced from the CMV promoter embedded upstream of the virus gene coding region in a run-off transcription reaction.
  • Flaviviruses are positive-sense and their genomic RNA can serve directly as message for translation of proteins required for virus replication, this reaction produces chimeric DEN2/RBV RNA transcripts that are infectious and are capable of producing progeny virions in host cells.
  • the larger pr segment is released into the extracellular medium, although prM/M cleavage is sometimes incomplete so that some prM remains in virions.
  • Antibodies to prM may be undesirable in the case of DEN, since they can play a role in immune enhancement. For that reason, construction of a chimeric virus containing only DEN E protein can also be performed. The E-only construct is used if growth is not inferior to the alternative prM-E chimera.
  • each virus particle contains a multi-copy presentation of 180 copies of the E protein with identical amino acid sequence assembled in a dimeric head to tail configuration.
  • the E protein copies contain both type-specific and cross- reactive DEN neutralizing epitopes required to elicit protective immunity.
  • the NS proteins in this case encoded by the RBV vector remain either in the cell or (if soluble and released, like NS1), are removed during vaccine purification. Once the chimeric genome RNA is available, cells are transfected for preparation of a Research Master Seed (P2 after transfection).
  • Chimerization is known to attenuate virulence of Flaviviruses and may or may not affect replication in vitro.
  • the virus is adapted by 10 sequential passages. This passage series also assesses genetic stability of the chimera. Growth kinetics are used to compare the unpassaged and P10 virus; it is expected that approximately 0.5 logio higher yields will result (e.g., See Figure 3).
  • Sequencing will be performed to determine the mutation(s) associated with enhanced growth and these are introduced into the infectious clone by site-directed mutagenesis.
  • a new Research Virus Seed (P10+) is manufactured. Growth kinetics at MOI 0.1 to 0.001 are evaluated and the optimal MOI selected.
  • the seed virus is used to infect cells grown in stationary cultures (T 225 flasks), and culture fluid harvested and partially purified by removing cell debris (0.8 ⁇ filter), concentrating the virus by ultrafiltration/diafiltration (100 kDa filter), and inactivating the virus with 0.1% -propiolactone or another suitable method of inactivation.
  • Absence of residual live virus will be determined by inoculating Vero indicator cells (2 blind passages, 7 days apart) and performing plaque assays (by immunohistochemistry) on cell culture supernate.
  • Vaccine antigen potency is determined by dengue-specific monoclonal ELISA standardized to the plaque assay for live virus. Vaccine is frozen and formulated with adjuvants immediately prior to administration to mice.
  • Donor DEN 1 , 3 and 4 strains are selected based on high growth in Vero (or another cell substrate selected as described). Similar cloning templates to the ones used to generate a high-yield DEN2 (prM-E)/RBV chimera are used to construct chimeras containing DEN 1 , 3 and 4 prM-E (or E gene if viable for DEN2/RB V), with strict observance of gene junction sequences, particularly the prM signal sequence. PvBV genomic sequences are propagated in one plasmid ( Figure 7A).
  • each construct and growth kinetics in the selected cells for manufacture is defined, and if necessary, adaptation for high growth may be performed as described above for the P1 - P10 adaptation of RBV.
  • This work generally proceeds in a predictable manner, based on experience with the DEN 2/RBV construct.
  • Growth is optimized to ensure that the viruses generated can be used for manufacturing by (i) adaptation (serial passage) and/or (ii) inserting mutations (by site directed mutagenesis of the infectious clone) in the RBV vector backbone known to be associated with higher growth. These mutations may include those shown in Tables 3 and 4 in the nonstructural or 3 '-NCR of Rio Bravo virus. Research Seed Viruses are made and potency (PFU/mL) and identity confirmed by sequencing.
  • inactivated vaccine The methods for production of inactivated vaccine are those well known in the art, and have been described in patents and patent applications, and various publications (e.g., WO/2006/122964; Srivastava et al, 2001, Vaccine 19: 4557-65). Briefly, cells grown in suspension, on microcarrier beads, or on the surface of roller bottles or cell factories are infected with working seed virus at the appropriate MOI. Virus is harvested from cell cultures at the appropriate time after infection determined by growth curve studies, generally when CPE is just beginning. For example, the virus harvest is clarified by passage through a depth filter, digested with Benzonase® (nuclease) and ultrafiltered/diafiltered to remove host DNA.
  • Benzonase® nuclease
  • RBV chimeric den/RBV
  • Additional purification steps can employ methods well known in the art including protamine sulfate precipitation, cellufme sulfate chromatography, anion-exchange and size exclusion chromatography, and sucrose gradient centrifugation. Inactivation of the virus is achieved either before, between steps, or after purification.
  • Methods that can be used to inactivate the dengue/Rio Bravo or dengue/Uganda S subgroup chimeric viruses include treatment with chemicals (such as, but not limited to formalin, beta-propiolactone, aziridines (e.g. ethyleneimine), hydrogen peroxide, organic solvents, and ascorbic acid), high pressure, ultraviolet radiation with or without psoralen sensitization or gamma irradiation. These methods invariably degrade the viral RNA by alkylation or other reactions. In contrast, the dengue-mosquito Flavivirus chimera is incapable of replication in mammalian cells and can be used without chemical or other means of inactivation, preserving the structure of the viral RNA.
  • chemicals such as, but not limited to formalin, beta-propiolactone, aziridines (e.g. ethyleneimine), hydrogen peroxide, organic solvents, and ascorbic acid
  • UV radiation with or without psoralen sensitization or gamma irradiation.
  • Application of these methods yields a purified, inactivated dengue vaccine with a potency of greater than or equal to 10 9 PFU in the form of inactivated virions per 0.5 mL of vaccine, 0.5 mL representing the volume typically injected into humans.
  • the method will yield a potency of 10 10 PFU in the form of inactivated virions per 0.5 mL.
  • the equivalent amount of viral envelope protein in the formulation expressed in micrograms is greater or equal to 10 micrograms/0.5 mL dose.
  • the method will yield a potency of 20 micrograms of viral protein, or greater/0.5 mL.
  • a potency in the range of 10 9 whole virions/mL or 10 micrograms is not reproducibly achievable by standard methods of propagating dengue virus itself and potencies of 10 10 whole virions or 20 micrograms per human dose cannot be achieved by standard methods.
  • Similar methods of producing chimeric inactivated vaccines can be applied to the manufacture of high-titer inactivated vaccines with a potency of >10 9 PFU (equivalent as inactivated virus) or >10 micrograms of protein per 0.5mL against other Flaviviruses that are difficult to propagate to high titer in cell cultures, such as yellow fever 17D virus.

Abstract

L'invention concerne des vecteurs de flavivirus chimérique codant pour une ou plusieurs protéines structurales d'un premier flavivirus avec un faible niveau de réplication dans une cellule, tel que le virus de la dengue et le virus de la fièvre jaune, et une structure centrale d'un second flavivirus avec un niveau élevé de réplication dans la cellule, tel que le virus Rio Bravo ou le virus Ouganda S. Les flavivirus chimériques codés par les vecteurs de flavivirus chimérique de l'invention peuvent être utilisés pour vacciner des sujets pour prévenir une infection par des flavivirus infectieux, y compris les virus de la dengue et les virus de fièvre jaune.
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